http://2009.igem.org/wiki/index.php?title=Special:Contributions/Frans&feed=atom&limit=50&target=Frans&year=&month=2009.igem.org - User contributions [en]2024-03-29T12:58:51ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T22:24:26Z<p>Frans: </p>
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<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
[[Image:UptakeRPU.png]] <br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
[[Image:Uptake100um.png]] <br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter test, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100uM As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD measurements of the growing cell measurements showed that concentrations as high as 5000&micro;M Arsenite are poisonous for E.Coli top 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
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{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T21:48:32Z<p>Frans: /* Export of arsenicum via Ars operon */</p>
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<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Application}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
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<div class="intro"><br />
<h1>Transport</h1><br />
<b>To isolate heavy metals from the environment we require uptake systems. Several different mechanisms to create such an import system are exist; metal transporters (coupled and uncoupled) and binding proteins in the periplasm. Import systems for several metals were found. We investigated HmtA for copper/zinc uptake. Cloning of the HmtA failed unfortunatly. Citrate coupled transporters, CitH and CitM were also considered as wel as the periplasmic accumulation operon Mer.<br />
Since we chose to focus on arsenic the final device was made with GlpF. GlpF a aquaglycerol porin was found to import not only glycerol but also arsenite and arsenate. This importer was cloned as a BioBrick part and transformed into ''E. coli''. An uptake assay was performed and a metal sensitivity assay, which showed functionality of the GlpF transporter.</b><br />
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</div><br />
|}<br />
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<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of ''E. coli'' is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]].<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
<br />
[[Image:DeathAssayWT.png|310px|left]]<br />
[[Image:DeathAssayGlpF.png|310px|left]]<br />
[[Image:DeathAssayLow.png|310px|left]]<br />
<br />
The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is. <br />
<br />
The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <br />
<br />
In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD<sub>600</sub> 0.9. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.<br />
<br />
==={{anchor|Modelling}}Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
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.infoIcon { display: inline; }<br />
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The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
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<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
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<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
===Export of arsenicum via Ars operon===<br />
<br />
GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
<br />
[[Image:ArsRBC_operon.PNG|600px]]<br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in ''E. coli'' ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T21:47:36Z<p>Frans: /* {{anchor|Modelling}}Modelling uptake GlpF */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Application}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
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{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Transport</h1><br />
<b>To isolate heavy metals from the environment we require uptake systems. Several different mechanisms to create such an import system are exist; metal transporters (coupled and uncoupled) and binding proteins in the periplasm. Import systems for several metals were found. We investigated HmtA for copper/zinc uptake. Cloning of the HmtA failed unfortunatly. Citrate coupled transporters, CitH and CitM were also considered as wel as the periplasmic accumulation operon Mer.<br />
Since we chose to focus on arsenic the final device was made with GlpF. GlpF a aquaglycerol porin was found to import not only glycerol but also arsenite and arsenate. This importer was cloned as a BioBrick part and transformed into ''E. coli''. An uptake assay was performed and a metal sensitivity assay, which showed functionality of the GlpF transporter.</b><br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of ''E. coli'' is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]].<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
<br />
[[Image:DeathAssayWT.png|310px|left]]<br />
[[Image:DeathAssayGlpF.png|310px|left]]<br />
[[Image:DeathAssayLow.png|310px|left]]<br />
<br />
The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is. <br />
<br />
The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <br />
<br />
In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD<sub>600</sub> 0.9. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.<br />
<br />
==={{anchor|Modelling}}Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<html><style type="text/css"></html><br />
{{InfoBox/Style.css}}<br />
.infoIcon { display: inline; }<br />
<html></style></html><br />
The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
==Export of arsenicum via Ars operon==<br />
<br />
GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
<br />
[[Image:ArsRBC_operon.PNG|600px]]<br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in ''E. coli'' ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T21:46:38Z<p>Frans: /* Export of arsenicum via Ars operon */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Application}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Transport</h1><br />
<b>To isolate heavy metals from the environment we require uptake systems. Several different mechanisms to create such an import system are exist; metal transporters (coupled and uncoupled) and binding proteins in the periplasm. Import systems for several metals were found. We investigated HmtA for copper/zinc uptake. Cloning of the HmtA failed unfortunatly. Citrate coupled transporters, CitH and CitM were also considered as wel as the periplasmic accumulation operon Mer.<br />
Since we chose to focus on arsenic the final device was made with GlpF. GlpF a aquaglycerol porin was found to import not only glycerol but also arsenite and arsenate. This importer was cloned as a BioBrick part and transformed into ''E. coli''. An uptake assay was performed and a metal sensitivity assay, which showed functionality of the GlpF transporter.</b><br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of ''E. coli'' is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]].<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
<br />
[[Image:DeathAssayWT.png|310px|left]]<br />
[[Image:DeathAssayGlpF.png|310px|left]]<br />
[[Image:DeathAssayLow.png|310px|left]]<br />
<br />
The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is. <br />
<br />
The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <br />
<br />
In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD<sub>600</sub> 0.9. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.<br />
<br />
==={{anchor|Modelling}}Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<html><style type="text/css"></html><br />
{{InfoBox/Style.css}}<br />
.infoIcon { display: inline; }<br />
<html></style></html><br />
The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
<br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in ''E. coli'' ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T21:44:53Z<p>Frans: /* Planning and requirements */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Application}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Transport</h1><br />
<b>To isolate heavy metals from the environment we require uptake systems. Several different mechanisms to create such an import system are exist; metal transporters (coupled and uncoupled) and binding proteins in the periplasm. Import systems for several metals were found. We investigated HmtA for copper/zinc uptake. Cloning of the HmtA failed unfortunatly. Citrate coupled transporters, CitH and CitM were also considered as wel as the periplasmic accumulation operon Mer.<br />
Since we chose to focus on arsenic the final device was made with GlpF. GlpF a aquaglycerol porin was found to import not only glycerol but also arsenite and arsenate. This importer was cloned as a BioBrick part and transformed into ''E. coli''. An uptake assay was performed and a metal sensitivity assay, which showed functionality of the GlpF transporter.</b><br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of ''E. coli'' is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]].<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
<br />
[[Image:DeathAssayWT.png|310px|left]]<br />
[[Image:DeathAssayGlpF.png|310px|left]]<br />
[[Image:DeathAssayLow.png|310px|left]]<br />
<br />
The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is. <br />
<br />
The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <br />
<br />
In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD<sub>600</sub> 0.9. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.<br />
<br />
==={{anchor|Modelling}}Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<html><style type="text/css"></html><br />
{{InfoBox/Style.css}}<br />
.infoIcon { display: inline; }<br />
<html></style></html><br />
The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
<br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in ''E. coli'' ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
<br />
<br />
<br />
<br />
==Export of arsenicum via Ars operon==<br />
<br />
GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
<br />
[[Image:ArsRBC_operon.PNG|600px]]<br />
<br />
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{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project_PlanTeam:Groningen/Project Plan2009-10-21T21:43:51Z<p>Frans: /* {{anchor|Deliverables}} Project Deliverables */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Project Plan]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Project Plan]]<br />
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<br />
'''Judges:''' Please note [[Team:Groningen/Project_Plan#UPEDU|this section]] in particular.<br />
<br />
The project plan (known in [http://www.upedu.org/ UPEDU] as "[http://www.upedu.org/upedu/process/artifact/ar_sdp.htm Software Development Plan]") is meant to hold all information necessary for the management of the project. This includes things like the planning, role assignments, information on resources, etc. This particular project plan applies to the 2009 iGEM project at the University of Groningen.<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Parts/Used_Parts}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project_Plan#UPEDU}}</div><br />
<br />
=={{anchor|ProjectOverview}} Project Overview==<br />
===Project Purpose, Scope, and Objectives===<br />
The purpose of the 2009 iGEM Groningen project is [https://2009.igem.org/Judging/Judging_Criteria to have a great summer, and have fun attending the Jamboree].<br />
<br />
===Assumptions and Constraints===<br />
This plan assumes that we can raise enough funds to acquire all the needed materials, and that the university will supply the necessary facilities (like lab space). These will have to be organized before the start of the summer, as both iGEM and our individual schedules require us to do most of the work during the summer. We assume that we can collect enough data during the summer labwork and modelling to be presented during the iGEM Jamboree in oct/nov 2009. <br />
<br />
Our assumptions and constraints were defined in a list of wishes and demands as followed:<br />
<br />
<b><u>Demands</u></b><br />
:*Labwork is feasible in 3 month with 8 students.<br />
:*Modelling feasible in 3 months with 3 students.<br />
:*Everyone has to agree with the idea<br />
:*Financially feasible with a budget of 31.170,00 euro of which 8000 euros used for labwork.<br />
:*A new concept, not yet done before in this way, either the iGEM or in the synthetic biology<br />
:*Meets the criteria of iGEM<br />
:*Materials have to be attainable, either via the RuG or able to order in.<br />
:*Parameters for modelling have to be known, or available or attainable.<br />
:*Knowledge about the genes that we are using has to be available.<br />
<br />
<b><u>Wishes</u></b><br />
:*Not too complicated, not too many genes or gene clusters<br />
:*Has to have an application<br />
:*Using BioBricks that are easy to obtain, preferable available at the RuG.<br />
:*Preferably knowledge on the host, genes and/or end products has to be available at the RuG<br />
:*Some students in the team have experience working with the host.<br />
:*It is possible to find sponsoring for this project.<br />
<br />
==={{anchor|Deliverables}} Project Deliverables===<br />
During the course of this project the following will be delivered:<br />
<br />
*An initial idea for a new "machine" in the iGEM sense, including a preliminary feasibility study.<br />
*A detailed specification of the requirements of this machine.<br />
*A detailed design of this "machine", including an analysis of the usage scenarios and results of simulations of computer models of (parts of) this "machine".<br />
*The actual "machine" (consisting of cells)<br />
*A presentation for the jamboree in November.<br />
*A poster for the jamboree in November<br />
*A report of our findings<br />
<br />
====Planning and requirements for transporters====<br />
<br />
* '''Modelling:'''<br />
** Import speed<br />
** Amount <br />
** Max<br />
* '''Lab:'''<br />
** HmtA<br />
*** Zn/Cu alone<br />
*** B-type ATPase (could be use if there is a ATP shortage?)<br />
** CitM (probably not used)<br />
*** Divalent ions<br />
*** Citrate around<br />
*** Citrate can bind metals that are already bound.<br />
** Measurements (both for the "normal" cell and the cell with overexpression of the transporter)<br />
*** Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.<br />
*** Measure concentration of metal. difference between begin and end concentrations of metal outside the cell.<br />
*** How fast does it transport metal in/out the cell.<br />
**** Set up tests with (initial) extracellular concentrations of about <sup>1</sup>/<sub>3</sub>K (25% of V<sub>max</sub>), K (50% of V<sub>max</sub>), 3K (75% of V<sub>max</sub>) and 10mM (99.7% of V<sub>max</sub>, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V<sub>max</sub> at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.<br />
**** Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)<br />
**** This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).<br />
**** By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)<sub>ex</sub> (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.<br />
**** '''NOTE:''' Interestingly [[Team:Groningen/Literature#Kostal2004|Kostal 2004]] already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).<br />
<br />
===Evolution of the Project Plan===<br />
The project plan will be set up during the [[Team:Groningen/Project_Plan/Inception|Inception]] phase and after each iteration the [[#ProjectPlan|planning]] is updated to reflect the actual progress and give updated estimates. Other parts of the project plan are not scheduled to be updated after the Inception and will only be updated to fix errors, clarify the existing text or to reflect a change in circumstances.<br />
<br />
==Project Organization==<br />
This is covered by our [[Team:Groningen/Team|team page]] and Google Docs for contact information (for privacy reasons).<br />
<br />
=={{anchor|ManagementProcess}} Management Process==<br />
===Project Estimates===<br />
The estimated costs for the project are as presented in the balance sheet (GoogleDocs). The estimated time for the project is 3-4 hours a week per person from march-july. Full-time (about 40 hours a week per person) during the summer (july-september). From september untill the jamboree on 30 okt another 3-4 hours a week per person (turned out to be a lot more full-time for most people).<br />
<br />
==={{anchor|ProjectPlan}} Project Plan===<br />
This project recognizes the following [http://www.upedu.org/upedu/process/itrwkfls/iwf_iwfs.htm phases and milestones] (only broad descriptions of the milestones are given here):<br />
<br />
*[[Team:Groningen/Project_Plan/Inception|Inception]]: until 18 may, milestone: initial idea, including preliminary feasibility study.<br />
*[[Team:Groningen/Project_Plan/Elaboration|Elaboration]]: until the summer (1st of july), milestone: initial design and modelling.<br />
*[[Team:Groningen/Project_Plan/Construction|Construction]]: during the summer (1 july-30 august), milestone: the actual "product".<br />
*[[Team:Groningen/Project_Plan/Transition|Transition]]: after the summer (30 august-31 dec), milestone: the jamboree presentation and documentation for the next team.<br />
<br />
Note that during the elaboration there should probably already be some lab work (if at all possible) to assist in modelling, and during the summer the modelling work will probably continue. Detailed planning of each of the iterations should be documented in iteration plans.<br />
<br />
{| border="1"<br />
!Iteration<br />
!End date<br />
!Objectives<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/1|Inception 1]]<br />
|2009-04-20<br />
|Narrow down ideas to about three ideas that should be investigated further.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/2|Inception 2]]<br />
|2009-05-18<br />
|Choose idea we're going to work on. The most important requirements should be identified.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/1|Elaboration 1]]<br />
|2009-06-01<br />
|The initial design should be made, the requirements refined. Some initial model prototypes should be made to gain experience in modelling.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/2|Elaboration 2]]<br />
|2009-06-30<br />
|Models of our design should be made and verified, the design refined. And if possible some things may have to be verified and or tried out in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/1|Construction 1]]<br />
|2009-07-21<br />
|All necessary equipment and materials should be known and in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/2|Construction 2]]<br />
|2009-08-11 <br />
|A checkup if the system works, including all parts, should be able to be done.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/3|Construction 3]]<br />
|2009-09-01<br />
|Everything (in the lab) finished :)<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/1|Transition 1]]<br />
|15-10-09<br />
|Presentation for the jamboree and the wiki should be (made and) polished.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/2|Transition 2]]<br />
|31-12-09<br />
|Our documentation should be prepared for and transferred to the next team.<br />
|}<br />
<br />
===Project Monitoring and Control===<br />
<br />
====Requirements Management====<br />
The requirements for this system are captured in [[Team:Groningen/Vision|the Vision document]].<br />
<br />
====Quality Control====<br />
The '''Quality of the constructs''' developed by Labworkers should be conform the following quality control regulations<br />
<br />
After transformation of the cells with Your Favorite Construct:<br />
*Pick a few colonies.<br />
**Grow o/n culture of a single colony.<br />
**Continue with miniprep on o/n culture.<br />
**Check insert length by PCR and restriction analysis, as written in document: Quality control (Overdrachts document iGEM 2008).<br />
***For restriction analysis try to find a restriction enzyme which makes a single cut in the vector and a single cut in the insert, or a double digestion with one which cuts in the vector and one which cuts in the insert.<br />
*Streak positive colony with inoculation eye out for single colonies.<br />
**Pick a single colony.<br />
**Grow o/n culture and make glycerol stock (put -80 position on [https://2009.igem.org/Team:Groningen/Parts Parts List]) next day. <br />
**Miniprep and continue with cloning / checking insert length.<br />
<br />
====Reporting and Measurement====<br />
During the non-summer period (march-july, sept-oct) a meeting will be held on a weekly basis. During the meeting the progression of the project, finance, contact with other iGEM teams and organisation subjects will be discussed. Every other week the advisors are invited for this meeting. During the summer period (july-sept) a meeting will also be held on a weekly basis with the whole team.<br />
<br />
====Risk Management ====<br />
Risks will be identified in Inception Phase using the steps identified in the RUP for Small Projects activity “Identify and Assess Risks”. Project risk is evaluated at least once per iteration and documented in this table (See [[Team:Groningen/Project_Plan/Risk_List|Risk List]]).<br />
<br />
===={{anchor|ConfigurationManagement}} Configuration Management====<br />
See [[Team:Groningen/Project_Plan/Tools and Documentation|Tools and Documentation]].<br />
<br />
<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project_Plan/Risk_List}}</div><br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project_Plan}}</div><br />
=={{anchor|Process_Guidelines}}{{anchor|UPEDU}} Process Guidelines==<br />
This project attempts to follow a standardized process for software development ([http://www.upedu.org/ UPEDU]), adapting it for use with iGEM. Where possible roles, activities, etc. are simply copied from UPEDU (and ultimately RUP). To cope with the difference between software development and genetic engineering we make the following changes:<br />
<br />
*A new role "[[:Category:Team:Groningen/Roles/Modeller|Modeller]]" for someone who models the design on a computer and attempts to verify the design before it is implemented in the lab.<br />
*The "[[:Category:Team:Groningen/Roles/Implementer|Implementer]]" role is changed to reflect lab work instead of programming.<br />
*We also added some roles to take care of things like keeping minutes, doing PR and so on.<br />
<br />
In practice we ignored most of UPEDU, because of a lack of experience with such approaches within the team (and a lack of time to make up for it), making it inefficient to use, but also because some features proved hard to implement, we specifically had trouble with:<br />
<br />
*'''Using iterative development.''' In software development it is possible to make small changes quickly, allowing iterations as short as one or two weeks (much longer iterations would not have suited the time scale of iGEM). However, in synthetic biology it is more efficient to work on many things at once over a longer time frame (simply because each small change necessarily takes a certain amount of time). In practice this meant that a more waterfall-esque development process was used, where the construction phase consisted mostly of preparing everything for testing and analysis, leaving all testing, analysis and combination of results for the transition phase.<br />
*'''Making a clear separation between requirements(, architecture), design, implementation and tests.''' With software it is possible to create pretty much anything you can define, so one starts by defining what the system should do (at least in part), then figures out how this could be implemented in an abstract way and only then actually implements it (tests can usually be developed in parallel). In part this approach can and should(!) be adopted in synthetic biology, but some things do make it hard to do this rigorously. For example, there is hardly any standard way to abstractly talk about system components yet. Hopefully parts and "devices" might help with this in the near future.<br />
<br />
What did work for us to varying degrees:<br />
<br />
*'''Identifying risks''' early on in the project. Without UPEDU we probably would not have done this and this could have led to severe problems. Luckily not that many of the risks actually materialized, but we did implement some of the mitigation strategies, leading to less nasty surprises. For example, we made an overview of everyone's availability early on in the project and tried to prevent having only one person work on a single part as much as possible.<br />
*'''Using a standardized [[Team:Groningen/methodischontwerpen|design process]].''' Not specifically UPEDU, but it fits well within the framework and it helped us to structure our project selection process.<br />
*'''Recognizing different phases''' in our development. This especially helped in the beginning by giving at least some structure to our planning and providing a vocabulary to talk about such things.<br />
*'''Using iterations.''' Although we had definite problems in our use of iterations they did give some structure to the construction phase for example.<br />
*Describing our '''Tools and Documentation'''. Although not always used consistently it did help us keep an overview what was stored where.<br />
*Having specific '''Roles'''. Not all roles were used (consistently), but it did provide us with at least some structure to deal with eleven team members (it is not easy to do this "free-style").<br />
<br />
=={{anchor|Annexes}} Annexes==<br />
During the inception phase ideas were generated and selected using a [[Team:Groningen/methodischontwerpen|design template]]. For selection of the ideas a [[Team:Groningen/Project_Plan#Assumptions_and_Constraints|list of demands and wishes]] was made.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T21:38:51Z<p>Frans: /* Missing information/To Do */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Application}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
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{| style="clear:both"<br />
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<div class="intro"><br />
<h1>Transport</h1><br />
<b>To isolate heavy metals from the environment we require uptake systems. Several different mechanisms to create such an import system are exist; metal transporters (coupled and uncoupled) and binding proteins in the periplasm. Import systems for several metals were found. We investigated HmtA for copper/zinc uptake. Cloning of the HmtA failed unfortunatly. Citrate coupled transporters, CitH and CitM were also considered as wel as the periplasmic accumulation operon Mer.<br />
Since we chose to focus on arsenic the final device was made with GlpF. GlpF a aquaglycerol porin was found to import not only glycerol but also arsenite and arsenate. This importer was cloned as a BioBrick part and transformed into ''E. coli''. An uptake assay was performed and a metal sensitivity assay, which showed functionality of the GlpF transporter.</b><br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of ''E. coli'' is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]].<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
<br />
[[Image:DeathAssayWT.png|310px|left]]<br />
[[Image:DeathAssayGlpF.png|310px|left]]<br />
[[Image:DeathAssayLow.png|310px|left]]<br />
<br />
The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is. <br />
<br />
The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <br />
<br />
In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD<sub>600</sub> 0.9. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.<br />
<br />
==={{anchor|Modelling}}Modelling uptake GlpF===<br />
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The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
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<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
<br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in ''E. coli'' ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
<br />
<br />
==Planning and requirements==<br />
<br />
* '''Modelling:'''<br />
** Import speed<br />
** Amount <br />
** Max<br />
* '''Lab:'''<br />
** HmtA<br />
*** Zn/Cu alone<br />
*** B-type ATPase (could be use if there is a ATP shortage?)<br />
** CitM (probably not used)<br />
*** Divalent ions<br />
*** Citrate around<br />
*** Citrate can bind metals that are already bound.<br />
** Measurements (both for the "normal" cell and the cell with overexpression of the transporter)<br />
*** Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.<br />
*** Measure concentration of metal. difference between begin and end concentrations of metal outside the cell.<br />
*** How fast does it transport metal in/out the cell.<br />
**** Set up tests with (initial) extracellular concentrations of about <sup>1</sup>/<sub>3</sub>K (25% of V<sub>max</sub>), K (50% of V<sub>max</sub>), 3K (75% of V<sub>max</sub>) and 10mM (99.7% of V<sub>max</sub>, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V<sub>max</sub> at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.<br />
**** Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)<br />
**** This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).<br />
**** By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)<sub>ex</sub> (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.<br />
**** '''NOTE:''' Interestingly [[Team:Groningen/Literature#Kostal2004|Kostal 2004]] already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).<br />
<br />
==Export of arsenicum via Ars operon==<br />
<br />
GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
<br />
[[Image:ArsRBC_operon.PNG|600px]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T19:49:28Z<p>Frans: /* Cloning strategy */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
[[Image:UptakeRPU.png]] <br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
[[Image:Uptake100um.png]] <br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter test, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100uM As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD measurements of the growing cell measurements showed that concentrations as high as 5000&micro;M Arsenite are poisonous for E.Coli top 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
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arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
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{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===conclusion===<br />
The Fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T19:43:09Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
[[Image:UptakeRPU.png]] <br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
[[Image:Uptake100um.png]] <br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter test, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100uM As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD measurements of the growing cell measurements showed that concentrations as high as 5000&micro;M Arsenite are poisonous for E.Coli top 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part:BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===conclusion===<br />
The Fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T19:36:43Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
[[Image:UptakeRPU.png]] <br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
[[Image:Uptake100um.png]] <br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter test, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100uM As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD measurements of the growing cell measurements showed that concentrations as high as 5000&micro;M Arsenite are poisonous for E.Coli top 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part:BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T19:26:17Z<p>Frans: /* Conclusion */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
[[Image:UptakeRPU.png]] <br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
[[Image:Uptake100um.png]] <br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter test, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100uM As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD measurements of the growing cell measurements showed that concentrations as high as 5000&micro;M Arsenite are poisonous for E.Coli top 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part:BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 > 5000 > 5 > 50 > 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:GroningenTeam:Groningen2009-10-21T19:18:23Z<p>Frans: </p>
<hr />
<div><!-- {{Team:Groningen/Header}} <br />
[[Category:Team:Groningen]] --><br />
<br />
[[Image:Title_groningen.png|975px|center]]<br />
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<h1>Heavy metal scavengers<!-- with a vertical gas drive--></h1><br />
Human health and the environment are endangered by heavy metal pollution in water and sediment. To battle this problem, a '''purification strategy''', in which arsenic, zinc and copper are removed from water and sediment, was developed. This strategy encompasses a biological device in which <i>E. coli</i> bacteria accumulate metal ions from solutions, after which they '''produce gas vesicles''' and '''start floating'''. This biological device consists of two integrated systems: one for metal uptake and storage, the other for metal induced buoyancy. The uptake and storage system consists of a [[Team:Groningen/Project/Transport|metal transporter]] and [[Team:Groningen/Project/Accumulation|metal binding proteins]] (to reduce toxicity and increase accumulation). The buoyancy system is made up of a [[Team:Groningen/Project/Promoters|metal induced promotor]] in front of a [[Team:Groningen/Project/Vesicle|gas vesicle gene cluster]]. The combination of both systems will enable the efficient cleaning of polluted water and sediment in a biological manner. <br />
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<li id="arsenic"></html>It all starts with [[Team:Groningen/Application|arsenic]] in solution.<html></li><br />
<li id="transport"></html>[[Team:Groningen/Project/Transport|Metal transport]]<html></li><br />
<li id="accumulation"></html>[[Team:Groningen/Project/Accumulation|Metal accumulation]]<html></li><br />
<li id="gas"></html>[[Team:Groningen/Project/Vesicle|Gas Vesicle]]<html></li><br />
<li id="promoter"></html>[[Team:Groningen/Project/Promoters|Metal sensitive promoters]]<html></li><br />
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*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*{{todo}} November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*{{todo}} <b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] and [http://whereis-beta.mit.edu/?mapterms=lobby%2013&zoom=15&lat=42.35993922977393&lng=-71.092529296875&open=object-13 Lobby 13] in Cambridge, MA</b><br />
*{{todo}} October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*{{todo}} October 23<sup>rd</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div> --><br />
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{{Team:Groningen/Footer_Main}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T19:10:13Z<p>Frans: /* E. coli */</p>
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<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
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<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
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<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coli_.28according_to_Kostal_et_al._2004.29|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
[[Image:UptakeRPU.png]] <br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
[[Image:Uptake100um.png]] <br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ‘’E. coli’’ with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===conclusion===<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
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The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
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<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
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function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part:BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 > 5000 > 5 > 50 > 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T18:56:49Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter units (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). <br />
<br />
luorescence change due to a change in the internal As(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===conclusion===<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 > 5000 > 5 > 50 > 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T18:55:02Z<p>Frans: /* E. coli */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter units (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). <br />
<br />
luorescence change due to a change in the internal As(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====Fluorscence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===conclusion===<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 > 5000 > 5 > 50 > 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. in figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pCueO RFP construct.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/File:Promoters-CueO-OD.pngFile:Promoters-CueO-OD.png2009-10-21T18:39:23Z<p>Frans: </p>
<hr />
<div></div>Franshttp://2009.igem.org/File:Promoters-CueO.pngFile:Promoters-CueO.png2009-10-21T18:39:02Z<p>Frans: </p>
<hr />
<div></div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T18:23:33Z<p>Frans: /* fluorecence of growing cells */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====fluorecence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000&micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with plasmid psb1AC3 containing the pArsR RFP construct.<br />
<br />
===conclusion===<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T18:14:26Z<p>Frans: /* fluorecence of growing cells */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====fluorecence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T18:12:50Z<p>Frans: /* fluorecence of growing cells */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
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</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====fluorecence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part:BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50&micro;M arsenite which is as high as 85% of the fluorescence from reference promotor Bba_J23101. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite.<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T17:53:11Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
=====fluorecence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite.<br />
<br />
The fluorescence in figure 4 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0&micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4C&deg;. Induction to a final concentration of 5000&micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000&micro;M is the poisoning of the cells with Arsenite. <br />
<br />
<br />
[[Image:Promoters-ArsR-OD.png]]<br />
:Figure 5: Shows the OD plotted against time of E.coli with psb1AC3 containing the pArsR RFP construct.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/File:Promoters-ArsR-OD.pngFile:Promoters-ArsR-OD.png2009-10-21T17:19:02Z<p>Frans: </p>
<hr />
<div></div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T15:58:51Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to conc of 5000,500,50,5 and 0 &micro;M sodium arsenite.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T15:43:55Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter units(RPU) with. <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000,500,50,5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.<br />
<br />
[[Image:Promoters-ArsR.png]]<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/File:Promoters-ArsR.pngFile:Promoters-ArsR.png2009-10-21T15:43:15Z<p>Frans: </p>
<hr />
<div></div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T14:53:28Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter units(RPU) with. <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T14:09:40Z<p>Frans: /* cloning strategy */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest. We take into consideration the following promoters:'''<br />
<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [https://2009.igem.org/Team:Groningen/Protocols| protocols]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter units(RPU) with. <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
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</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T13:45:38Z<p>Frans: /* E. coli */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
=Promotors=<br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [https://2009.igem.org/Team:Groningen/Protocols| protocols]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter units(RPU) with. <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2009-10-21T13:09:46Z<p>Frans: /* Fluorescence measurement */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
[[Category:Protocol]]<br />
[[Category:Escherichia coli]]<br />
=Protocols=<br />
{|cellpadding="2" cellspacing="2" border="0"<br />
|'''[[Team:Groningen/Protocols#Cloning|<u>Cloning</u>]]'''<br />
|<!--Space--><br />
|'''[[Team:Groningen/Protocols#Quality_control|<u>Quality control</u>]]'''<br />
|<!--Space--><br />
|'''[[Team:Groningen/Protocols#Measurements|<u>Measurements</u>]]''' <br />
|<!--Space--><br />
|'''[[Team:Groningen/Protocols#List_of_solutions|<u>List of solutions</u>]]'''<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#PCR|PCR]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Colony_PCR|Colony PCR]]<br />
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|[[Team:Groningen/Protocols#Fermentation|Fermentation]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Media|Media]]<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Plasmid_Isolation|Plasmid Isolation]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Restriction_analysis|Restriction analysis]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Buoyancy_test|Buoyancy test]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Antibiotics|Antibiotics]]<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Restriction|Restriction]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Membrane_protein_isolation|Membrane protein isolation]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coli_.28according_to_Kostal_et_al._2004.29|Metal uptake assay for <i>E. coli</i>]] <br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Chemicals|Chemicals]]<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Annealing_synthetic_oligo.E2.80.99s|Annealing synthetic oligo's]]<br />
|<!--Space--><br />
|<!--Quality control--><br />
|<!--Space--><br />
|Fluorescence measurement<br />
|<!--Space--><br />
|<!--List of solutions--><br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Ligation|Ligation]]<br />
|<!--Space--><br />
|<!--Quality control--><br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Death_assay|Death assay]]<br />
|<!--Space--><br />
|<!--List of solutions--><br />
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|-<br />
|[[Team:Groningen/Protocols#Making_competent_cells|Making competent cells]]<br />
|<!--Space--><br />
|<!--Quality control--><br />
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|<!--Measurements--><br />
|<!--Space--><br />
|<!--List of solutions--><br />
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|-<br />
|[[Team:Groningen/Protocols#Transformation|Transformation]]<br />
|<!--Space--><br />
|<!--Quality control--><br />
|<!--Space--><br />
|<!--Measurements--><br />
|<!--Space--><br />
|<!--List of solutions--><br />
|<!--Space--><br />
|}<br />
<br />
==Cloning==<br />
===[http://openwetware.org/wiki/PCR PCR]===<br />
{|cellpadding="2" cellspacing="2" border="0"<br />
|12.5 μL Phusion mastermix<sup>*</sup><BR><br />
1 μL forward primer<BR><br />
1 μL reverse primer<BR><br />
0.5 μL template<BR><br />
10 μL demi water<BR><BR><br />
<sup>*</sup>Phusion master mix contains:<BR><br />
200 μL 5x Phusion HF buffer<BR><br />
8 μL 25 mM dNTP's<BR><br />
282 μL MilliQ water<BR><br />
10 μL Phusion Polymerase<br />
|<!--Space--><br />
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|<u><b>PCR Reaction</b><BR><br />
</u>Hotstart<BR><br />
95 °C, 2 min.<BR><br />
25 cycles<BR><br />
95 °C, 30 sec.<BR><br />
61 °C, 20 sec.<BR><br />
72 °C, 1.5 min.<BR><br />
End<BR>72 °C, 10 min.<br />
<BR>4 °C, ∞<br />
|}<br />
<br />
===Plasmid isolation===<br />
Usually performed using Miniprep kits like NucleoSpin<SUP><FONT SIZE="-1">®</FONT></SUP> Plasmid, (Machery nagel) or GeneElute™ Plasmid Miniprep Kit (Sigma-Aldrich). This is a consensus protocol.<br />
*Spin down ON Culture in table top centrifuge, 1 min. 13.000 RPM<br />
*Resuspend pellet, RNAse is added to degrade RNA (200 μL)<br />
*Add lysis buffer (200 μL), to lyse the cells and to release their contents<br />
*Add neutralization buffer (350 μL), proteins will denaturate<br />
*Centrifuge in table top centrifuge 10 min. 13.000 RPM<br />
*Add clear lysate in column provided in kit<br />
*Spin down 1 min. 13.000 RPM<br />
*Add wash buffer (usually needs EtOH to be added!) (500 - 750 μL)<br />
*Remove flow-through and spin again to remove residual wash buffer<br />
*Put column in clean 1.5 mL cup and add 15 - 50 μL MilliQ water or Tris buffer (pH=8.0)<sup>*</sup><br />
*Incubate for 1 - 2 min.<br />
*Spin down 1 min. 13.000 RPM<br />
<sup>*</sup>Less volume gives higher concentrations, supplied Tris buffer is claimed to give higher yields<br />
<br />
===Restriction===<br />
*Mix<br />
**1 μL 10x fast digest buffer ([http://www.fermentas.com/ Fermentas]) or correct [http://fermentas.com/techinfo/re/5bufferplussystem.htm#Buffers conventional buffer]<br />
**0.5 μL Enzyme A<sup>*</sup><br />
**0.5 μL Enzyme B<sup>*</sup><br />
**8 μL DNA to be digested<sup>**</sup><br />
*Incubate 0.5 - 1 h @ 37 °C (Fast digest do not require long incubations, when using conventional enzymes 1 h. should be maintained)<br />
*Purify cut plasmid using PCR clean up kit<br />
<sup>*</sup> a combination of two of the following (when using biobrick standard) [http://www.fermentas.com/catalog/re/fastbcui.htm <i>Spe</i>I], [http://www.fermentas.com/catalog/re/fastecori.htm <i>Eco</i>RI], [http://www.fermentas.com/catalog/re/fastpsti.htm <i>Pst</i>I] and/or [http://www.fermentas.com/catalog/re/fastxbai.htm <i>Xba</i>I]<br />
<br><sup>**</sup> When digesting vectors, bring the digested vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit ([http://www.mn-net.com/Products/NucleicAcidPurification/DNAcleanup/NucleoSpinExtractII/tabid/1452/language/en-US/Default.aspx NucleoSpin<SUP><FONT SIZE="-1">®</FONT></SUP> Extract II, Machery nagel], [http://www.zymoresearch.com/content/zymoclean-gel-dna-recovery-kit-d4001-d4002-d4007-d4007-d4001s Zymoclean™ Gel DNA Recovery Kit] or similar) to an end volume indicated by the kit (End volume determines concentration, variations are possible)<br />
(alternatively, [http://openwetware.org/wiki/Phosphatase_treatment_of_linearized_vector Phosphatase treatment of linearized vector])<br />
<br />
===Annealing synthetic oligo’s===<br />
<u>Phosphorylation of 5' ends & hybridization<SUP><FONT SIZE="-1">[http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts[1]]</FONT></SUP></u> <br />
*Mix: <br />
**3 μL 100 µM (anti-)sense oligo <br />
**1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])<sup>**</sup><br />
**2 μL 10mM ATP <sup>**</sup><br />
**1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)] <br />
**3 μL MilliQ<br />
***(for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)<br />
*Incubate @ 37 °C for 1.5 hours. <br />
*Mix <br />
**10 μL Sense mixture<br />
**10 μL Anti-sense mixture<br />
**3 μL 0.5 M NaCl <br />
*Place in boiling water for 3 min., and allow the reaction to cool to room temperature.<br />
**Upon reaching room temperature add restricted vector (see for ratio [[Team:Groningen/Protocols#Ligation|Ligation]]<br />
**If kept at low temperature before ligation heat up the annealing mixture up to 65 °C for 1 min. to prevent the formation of multimers<br />
<sup>**</sup>Alternatively T4 DNA Ligase buffer can be used, already containing ATP<br />
===[http://openwetware.org/wiki/DNA_ligation Ligation]===<br />
*Mix<sup>*</sup><br />
**1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4dnaligase.htm T4 ligase buffer]<br />
**7.5 μL vector (purified from gel)<br />
**1 μL Insert<br />
**0.5 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4dnaligase.htm T4 ligase]<br />
*Incubate<br />
:* 1h RT<br />
:or<br />
:* ON @ 4 °C<br />
<sup>*</sup>This is a consensus, calculations should be performed to have the ligations be done in a 5:1 - 10:1 (Insert:Vector) mass ratio. <br />
<br />
===Making competent cells===<br />
Competent cells: [http://openwetware.org/wiki/TOP10_chemically_competent_cells TOP10] & [http://openwetware.org/wiki/E._coli_genotypes#DB3.1 DB3.1]<br />
*10 mL ON culture is used to inoculate [[Team:Groningen/Protocols#LB.28Agar.29|LB]], 100 μL ON culture per 20 mL<sup>*</sup><br />
*Cultures are grown @ 37 °C until an OD<sub>600</sub> of 0.2 ~ 0.3 is reached.<br />
*Cultures are spinned down 5 min. @ 4000 rpm, 4 °C<br />
*Supernatant is removed and pellet (per 20 mL culture) is resuspended in 5 mL chilled 0.1 M [[Team:Groningen/Protocols#0.1_M_CaCl2|CaCl<sub>2</sub>]]<br />
**Suspension is incubated on ice for 10 min. <br />
*Suspensions are spinned down 5 min. @ 4000 rpm, 4 °C<br />
# Supernatant is removed and pellet is resuspended in 1770 μL chilled 0.1 M [[Team:Groningen/Protocols#0.1_M_CaCl2|CaCl<sub>2</sub>]] and supplemented with 230 μL 87% glycerol prior to making aliquots.<br />
# Cells are divided in 50 μL aliquots<br />
# Cells are snapfrozen in liquid nitrogen and stored @ -80 °C<br />
<sup>*</sup> Cultures should be grown in the ratio 1:5 (medium:air), so 10 mL culture in a 50 mL greiner tube.<br />
<br />
===Transformation===<br />
*Add 10 uL of ligation mixture or 1 uL isolated plasmid to competent cell aliquot<br />
** <b>+ control</b>: 1 μL <partinfo>pSB3K3</partinfo> or <partinfo>pSB1AC3</partinfo> plasmid (no death gene!), <b>- control</b>: 1 μL MilliQ<sup>*</sup><br />
** Alternatively a single cut plasmid can be taken as a ligation control<br />
<sup>*</sup>Alternative - control: 1 μL [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] or [http://partsregistry.org/Part:pSB3K3 pSB3K3] carrying ccdB deathgene<br />
*Incubate on ice for 15 - 30 min.<br />
*Heatshock 45 sec. @ 42 °C or 5 min. 37 °C<br />
*Let cells relax on ice for 1 - 2 min.<br />
*Add [[Team:Groningen/Protocols#LB.28Agar.29|LB]] 200 μL (or 800 μL when spinning cells down, see below)<br />
*Incubate 37 °C, 250 RPM for 1 h<br />
*Plate out on [[Team:Groningen/Protocols#LB.28Agar.29|LB-agar]] + [[Team:Groningen/Protocols#Kanamycin|Kanamycin]] (30 μg/ml for [http://partsregistry.org/Part:pSB3K3 pSB3K3]) or [[Team:Groningen/Protocols#Ampicillin|Ampicillin]] (100 μg/mL for [http://partsregistry.org/Part:pSB1AC3 pSB1AC3])<br />
**Plate out 50 μL & 200 μL (or 100 μL after spinning down and resuspending cells) of cell suspension<br />
*Grow ON @ 37 °C<br />
<br><u>Checking transformations</u><br />
*See if - control is empty for functioning antibiotics and death gene<br />
*See how many colonies on + control for functioning competent cells<br />
*See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)<br />
*If enough transformants, inoculate 3 - 5 colonies in an ON culture<br />
**Alternatively perform colony PCR<br />
<br />
==Quality control==<br />
===Colony PCR===<br />
*Put colony in 1 μL MilliQ water<br />
*Put colony suspension in microwave for 1 min. 1000 W<br />
*Use this as DNA template<br />
*PCR reaction<br />
<center><br />
{|cellpadding="2" cellspacing="2" border="0"<br />
|21 μL Taq mastermix<sup>*</sup><BR><br />
1 μL forward primer<BR><br />
1 μL reverse primer<BR><br />
1 μL template<BR><br />
1 μL Taq polymerase<BR><BR><br />
<sup>*</sup>Taq master mix contains:<BR><br />
100 μL Taq NH<sub>4</sub><BR><br />
8 μL dNTP's<BR><br />
80 μL MgCl<BR><br />
652 μL MilliQ water<br />
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|<u><b>PCR Reaction<sup>**</sup></b><BR><br />
</u>Hotstart<BR><br />
95 °C, 2 min.<BR><br />
25 cycles<BR><br />
95 °C, 30 sec.<BR><br />
61 °C, 20 sec.<BR><br />
72 °C, 1.5 min.<BR><br />
End<BR>72 °C, 10 min.<br />
<BR>4 °C, ∞<br />
|}<br />
</center><br />
*Put PCR product on agarose gel<br />
<sup>**</sup> Indication, actual reaction program depends on primer set (Temperature of annealing) and the length of the template (Duration of elongation)<br />
<br />
===Restriction analysis===<br />
See also [[Team:Groningen/Protocols#Restriction| Restriction]], however in checking the presence of a multitude of bricks more diverse enzymes can be used. Also the incubation time can be shortened (Pour an agarose gel, wait for it to solidify and put reaction on gel) because it is not required that everything is cut.<br />
<br />
===Membrane protein isolation===<br />
*Use 20 mL of ON culture to start main culture in 1L [[Team:Groningen/Protocols#LB.28Agar.29|LB]] medium containing 50 μg/mL [[Team:Groningen/Protocols#Ampicillin|ampicillin]]<br />
**Incubate 37 °C, 250 RPM until OD<sub>600</sub> = 0.6, (approximately 2.5h of incubation, check the OD<sub>600</sub> every hour)<br />
**Add inducer (e.g. [Team:Groningen/Protocols#IPTG|IPTG]])<br />
**Incubate 1 h, 37 °C, 250 RPM<br />
*Culture Wash.<br />
**Cool culture on ice<br />
**Spin down culture @ 8000 rpm, 10 min, 4°C<br />
**Wash pellet with 40 ml ice-cold 50 mM KPi, pH 7.0<br />
**Spin down culture @ 8000 rpm, 10 min, 4°C<br />
**Resuspend pellet in 12 ml 50 mM KPi pH 7.0<br />
*Homogenization<br />
**Sonication on ice<br />
**9 cycles of 15 sec. sonication, 45 sec. rest<br />
*Separation fractions<br />
**Spin down @ 8000 rpm, 10 min, 4 °C<br />
**Collect supernatant <br />
**Spin down at 90 000 rpm, 25 min, 4 °C<br />
**Resuspend pellet in 1 ml of 50 mM KPi pH 7.0 + 1M NaCl<br />
**Spin down @ 80 000 rpm, 25 min, 4 °C<br />
*Solubilization<br />
**Resuspend in 950 μl of solubilization buffer (50 mM KPi pH 8.0 + 400 mM NaCl + 20% glycerol) and 50 μl of 10% DDM <br />
**Incubate in 4°C with shaking for 30 min.<br />
**Spin down @ 80 000 rpm, 25 min in 4°C<br />
**Collect the supernatant<br />
**(Supernatant can be stored at this stage)<br />
**Add Ni-NTA resin (30 μL [[Team:Groningen/Protocols#Buffer_A|buffer A]], 0.1% DDM (Bis(4-chlorophenyl)methane))<br />
**Incubate ON @ 4 °C<br />
**Spin down, 4 min. 3500 RPM<br />
**Remove supernatant<br />
**was resin with 1 mL [[Team:Groningen/Protocols#Buffer_B|buffer B]], 0.1% DDM<br />
**Spin down, 4 min. 3500 RPM<br />
**Remove supernatant<br />
**Elute protein by [[Team:Groningen/Protocols#Buffer_C|buffer C]], 0.1% DDM (50 μL)<br />
**Add protein loading buffer (with dithiothreitol (DTT))<br />
**Run 12% SDS-PAGE<br />
**Continue to Coomassie staining<br />
<br />
Staining of SDS-PAGE gels with Coomassie Brilliant Blue<br />
*Heat gel in staining solution and shake for 10 min.<br />
*Poor off staining solution and add destain.<br />
*Heat gel in destaining solution and shake.<br />
*Replace destaining solution after 10 min and repeat until ready.<br />
<br />
==Measurements==<br />
===Fermentation===<br />
Performed in 2L autoclavable fermenter with dished bottom vessel stirred fermenter<br />
*Autoclave closed fermenter system <br />
*Inoculate 1.3 L [[Team:Groningen/Protocols#LB.28Agar.29|LB]] (+100 µL [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A6457|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&lang=en_US%3E Y30 antifoam]) with 20 mL ON culture was used to <br />
**Airflow rate of 1 vvm <br />
**pH @ 7 (by addition of 4 M NaOH or 1 M HCl) <br />
**Temperature @ 37 °C<br />
**Agitation 400 to 800 RPM<sup>*</sup> <br />
**Oxygen concentration >50%<br />
*Take samples every 0.5 to 1 h. to determine optical density at 600 nm <br />
*50 mL samples in every growth phase (pre-exponential, early exponential, exponential, late exponential, steady state) <br />
*Spin samples down 35 min., 1000 RPM and remove supernatant<br />
*Continue [[Team:Groningen/Protocols#Buoyancy_test|Buoyancy test]]<br />
<br />
<sup>*</sup>6-bladed flat disc turbine (Rushton type) impeller (60 mm diameter) at the bottom to disperse the bubbles coming from the sparger underneath and a 3-bladed marine impeller, vortex (60 mm diameter) halfway the broth volume to create an axial flow.<br />
===Buoyancy test===<br />
Continued from cultures (flask or fermenter) after centrifugation<br />
*Resuspend pellet in 1 - 5 mL [[Team:Groningen/Protocols#Saline_solution_.280.15_M.2C_0.9.25_NaCl.29|saline solution]]<br />
*Determine OD<sub>600</sub> <br />
*Dilute suspension to OD<sub>600</sub> 1.5 with [[Team:Groningen/Protocols#Saline_solution_.280.15_M.2C_0.9.25_NaCl.29|saline solution]]<br />
*Put homogeneous suspension in tubes, take care of descent lighting from behind (day light is best)<br />
*Record decrease of buoyancy (matter of hours in fermentation cultures, days in shakeflask cultures)<br />
===Metal uptake assay for <i>E. coli</i><sup><FONT SIZE=-5>[[Team:Groningen/Literature#Kostal2004|Kostal 2004]]</FONT></sup>===<br />
*Grow ON culture of <i>E. coli</i> @ 30 °C <br />
**Use <i>E. coli</i> + control vector, <i>E. coli</i> + [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190023 pArsR]-[http://partsregistry.org/Part:BBa_E1010 RFP], <i>E. coli</i> + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT]<br />
*Inoculate day culture 1:50, grow in 1L [[Team:Groningen/Protocols#TB_medium|TB]]-[[Team:Groningen/Protocols#Ampicillin|Amp]] (100ml per time/[As(III)] sample)<br />
**Take OD<sub>600</sub> samples every 1 - 1.5 h of <i>E. coli</i> + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT]<br />
**Induce <i>E. coli</i> + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT] at OD<sub>600</sub> ~0.6 with 0.5 mM IPTG.<br />
*Harvest the cells @ stationary phase (after ~30 h) by spinning down @ 4000 RPM for 20 min. in Sorval centrifuge. <br />
*Wash 2 times with [[Team:Groningen/Protocols#TB74S_Buffer|TB74S buffer]]<br />
*Resuspend in prewarmed (30 °C) [[Team:Groningen/Protocols#TB74S_Buffer|TB74S buffer]] up to a OD<sub>600</sub> of ~25<br />
**Take a 1 mL sample in small aluminum boxes and dry @ 104 °C for >4 h<br />
**Afterwards measure the dry weight of the sample and calculate the weight/volume of the entire sample.<br />
*For the concentration range:<br />
**Incubate 5 samples (of same time point) for 1h @ 30 °C with 0μM, 10 μM, 20 μM, 50 μM and 100 μM As(III).<br />
*For the concentration range:<br />
**Incubate 5 samples (of same concentration) @ 30 °C with 10 or 100μM As(III) for 0, 10, 20, 40, 60 min.<br />
*Harvest cells by spinning down.<br />
*Wash the cells with [[Team:Groningen/Protocols#TB74S_Buffer|TB74S buffer]]<br />
*Resuspend in 10ml demi water.<br />
*Dry sample @ 65 °C for 2 days.<br />
*Store @ 4 °C or -80 °C<br />
*Determine the amount of As(III) in the cell at different stages and at different uptake concentrations using [http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry ICP-MS]<br />
<br />
====Analysis of arsenic concentration of ICP-MS====<br />
<br />
*Weigh 0.1g dried <i>E. coli</i> cells.<br />
*Add 5 ml 65% nitric acid.<br />
*For destruction the following microwave program was used:<br />
<BR><center><br />
{|cellpadding="1" cellspacing="1" border="1"<br />
|''' '''<br />
|'''Stage 1'''<br />
|'''Stage 2''' <br />
|-<br />
|Power(max) <br />
|1200 <br />
|1200<br />
|-<br />
|Power(%)<br />
|100 <br />
|100<br />
|-<br />
|Ramp(min) <br />
|15 <br />
|15<br />
|-<br />
|Hold(min) <br />
|0<br />
|30<br />
|-<br />
|Temp(°C) <br />
|140 <br />
|210<br />
|}</center><br />
*Let the samples cool down.<br />
*Dilute the samples by adding demi water up to 50 mL <br />
*If needed, spin down 15 min. @ 4000rpm in a Sorvall centrifuge.<br />
*Measure the arsenic concentration by [http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry ICP-MS] using both the standard mode (shows interference peak from multi-atomic molecule argon-chloride with the arsenic peak) and the collusion cell technology mode (doesn’t show the interference peak but has a 10x lower resolution than standard mode). <br />
**Use a standard curve between 0 - 10 µg As/L and 0 - 100 µg As/L using a certified 1000 ppm (mg/L) stock<br />
===Fluorescence measurement===<br />
<br />
A culture was made by colony picking from plate and grown in a reaction tube with 3 ml of LB and 100ug/ml ampiccilin overnight in a shaking incubator at 36 C. The cell suspensions were diluted 1:20 in fresh LB Amp 100 ug/ml and grown in a greiner tube of 50 ml in a shaking incubator at 36 C. The cells were grown to a OD600 of 0.5 and pelleted by centrifuging 4000g 10 min 4 C. The cells were resuspended in half of the volume with LB amp 100mg/ml and stored at 4 C for 30 min. After resuspension the cells were loaded onto a 96 well plate up to a volume of 250 ul per well. <br />
The cells were induced by adding 1,25 ul of the following stock solutions: <br />
<br />
1M, 100mM, 10mM, 1mM CuSO4 <br />
1M, 100mM, 10mM, 1mM ZnSO4<br />
1M ,100mM, 10mM, 1mM NaAsO2<br />
<br />
Creating the following end concentrations:<br />
5000uM, 500uM, 50uM, 5uM and 0uM<br />
<br />
The fluorescence and OD600 measurements were taken approximately every hour.<br />
The RFP was excited at 580 nm and emission was measured at 609 nm. <br />
Between the measurements the plates were stored in a shaking incubator at 36 C.<br />
<br />
===Death assay===<br />
Metal sensitivity assay<sup><FONT Style=-5>[[Team:Groningen/Literature#Lewinson2009|Lewinson 2009]]</FONT></sup> <br />
<br />
Measurement:<br />
<br />
*Grow selected strains ON in [[Team:Groningen/Protocols#LB.28Agar.29|LB medium]] with or without antibiotic<br />
*Induce in culture with inducer (in our case 0.5 mM [[Team:Groningen/Protocols#IPTG|IPTG]])<br />
<br />
Strains used in our tests<br />
{|border="1" <br />
!Test 1:<br />
!Test 2:<br />
|-<br />
|WT (+pSB1AC3) <br />
|WT (+pSB1AC3)<br />
|-<br />
|pLac-HmtA <br />
|pLac-HmtA<br />
|-<br />
|pLac-GlpF <br />
|pLac-GlpF<br />
|-<br />
|pLac-GlpF-fMT <br />
|pLac-GlpF-fMT<br />
|-<br />
|<br />
|plow-GlpF-fMT<br />
|-<br />
|<br />
|pLac-GlpF<br />
|-<br />
|}<br />
<br />
*Measure OD<sub>600</sub> of ON culture and dilute to an OD<sub>600</sub> of 0.05 in [[Team:Groningen/Protocols#LB.28Agar.29|LB]]+[[Team:Groningen/Protocols#Antibiotics|antibiotic]] & inducer ([[Team:Groningen/Protocols#IPTG|IPTG]] in our case). Inducer should be right concentration for use in microtiterplate (because you then dilute culture 150/200=1.33 times)<br />
*Add 150 ul of culture to 96 well microtiter plate (in triplo/quadruplo)<br />
*Add desired concentration of selected metal in 50 μL [[Team:Groningen/Protocols#LB.28Agar.29|LB]]+[[Team:Groningen/Protocols#Antibiotics|antibiotic]]<br />
<br />
Metals used in our test<br />
{|border="1" <br />
!Metal Concentration<br />
|-<br />
|NaAsO<sub>2</sub><br />
|0 μM <br />
|1 μM <br />
|10 μM <br />
|50 μM<br />
|-<br />
|CuSO<sub>4</sub> <br />
|0 μM <br />
|50 μM <br />
|250 μM <br />
|500 μM<br />
|}<br />
<br />
*Measure in Tecan Infinite 200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) or Tecan microplate *reader. Protocol:<br />
**Measure OD at 600 nm,<br />
**Every 15 minutes for 16-20hrs<br />
**Linear shaking, 6mm<br />
**37°C<br />
<br />
<br />
Analysis:<br />
<br />
*Plot for different strains OD<sub>600</sub> against time<br />
*Plot for different strains, the different metal concentrations against OD<sub>600</sub> at 12 hours<br />
<br />
=List of solutions=<br />
==Media==<br />
===LB(Agar)===<br />
*10 g (Bacto)Trypton<br />
*10 g NaCl<br />
*5 g Yeast extract<br />
*(1.5% Agar, 15 g)<br />
*Dissolve in 1 L demi water<br />
*Autoclave<br />
*Store @ RT (LB) or 60 °C (LB-Agar)<br />
<br />
===TB medium===<br />
* 12g Bacto-Tryptone<br />
* 24g Bacto-Yeast Extract<br />
* 4ml Glycerol [87%]<br />
* dissolve in 900ml demi water<br />
*Separetely prepare 100 mL Kpi <br />
**0.17M KH2PO4 (mw=136.09g/mol) (6.94g/300ml)<br />
**0.72M K2HPO4 (mw=174.18g/mol) (7.62g/300ml)<br />
**dissolve in demi water<br />
*Autoclave and mix<br />
<br />
==Antibiotics==<br />
===[http://openwetware.org/wiki/Ampicillin Ampicillin]===<br />
100 mg/ml Ampicillin (1000x) Stock<br />
* 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)<br />
* Add NaOH or KOH to allow the Ampicillin to dissolve<br />
* Filter sterilize 0.2 μm filter and aliquot<br />
* Store -20 °C<br />
===[http://openwetware.org/wiki/Chloramphenicol Chloramphenicol]===<br />
35 mg/ml Chloramphenicol (1000x) Stock<br />
* 0.35 g in 10 mL 100% EtOH<br />
* Filter sterilize 0.2 μm filter and aliquot<br />
* Store -20 °C<br />
===[http://openwetware.org/wiki/Kanamycin Kanamycin]===<br />
50 mg/ml Kanamycin (1000x) Stock<br />
* 500 mg in 10 mL demi water<br />
* Filter sterilize 0.2 μm filter and aliquot<br />
* Store -20 °C<br />
<br />
==Chemicals==<br />
===Buffer A===<br />
*10 mM Imidazole <br />
*600 mM NaCl<br />
*50 mM KPi pH 8.0<br />
*10% Glycerol<br />
*0.1% DDM<br />
*Demi water<br />
===Buffer B===<br />
*20 mM Imidazole <br />
*600 mM NaCl<br />
*50 mM KPi pH 8.0<br />
*10% Glycerol<br />
*0.1% DDM<br />
*Demi water<br />
===Buffer C===<br />
*500 mM Imidazole <br />
*600 mM NaCl<br />
*50 mM KPi pH 8.0<br />
*10% Glycerol<br />
*0.1% DDM<br />
*Demi water<br />
===0.1 M CaCl<sub>2</sub>=== <br />
*0.3319 g CaCl<sub>2</sub><br />
*Dissolve in 30 mL demi water<br />
===Destaining solution===<br />
*16 % methanol<br />
*10 % acetic acid<br />
*74 % water <br />
===0.15 M NaCl (Saline solution, 0.9% NaCl)===<br />
*9 g NaCl<br />
*Dissolve in 1 L demi water<br />
===4 M NaOH===<br />
*160 g NaOH<br />
*Dissolve in 1 L demi water<br />
===~1 M HCl===<br />
*500 mL demi water<br />
*500 mL HCl (37%, 11 M)<br />
===[http://openwetware.org/wiki/IPTG 1 M IPTG]===<br />
*2.38 g Isopropyl-beta-D-thiogalactopyranoside (IPTG) in 10 mL demi water.<br />
*Filter sterilize with a 0.22 μm syringe filter.<br />
*Store in 1 mL aliquots at -20 °C.<br />
<br />
===Sodium Arsenite (III)===<br />
*100mM Na-As solution <br />
*filter sterilize<br />
===Staining solution===<br />
*0.25 % Coomassie Brilliant Blue R-250<br />
*50 % methanol<br />
*10 % acetic acid<br />
*40 % water<br />
===TB74S Buffer===<br />
*0.605 g Tris (5mM)<br />
*8.76 g NaCl (150mM)<br />
*Dissolve in 1 L Demi water<br />
*Set pH with HCl to 7.4<br />
===[http://openwetware.org/wiki/TBE 10x TBE buffer]===<br />
*108 g Tris<br />
*55 g Boric acid<br />
*8.3 g EDTA<br />
*Dissolve in 1 L demi water<br />
*Adjust pH to 8.3<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-10-21T12:54:49Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
<div class="introduction"><br />
=Accumulation=<br />
Once heavy metals have entered the cell, it is crucial to keep them there. As these metals are toxic to cell survival in critical amounts, evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variety of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..</div><br />
<br />
=Metallothioneins=<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). The contain a conserved cys-x-cys or cys-x-his motif which coordinates metal binding, as can be seen in figure 1. They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity (Kb), they are ''in vivo'' used as a defense against oxidative stress by chelating metals. This proteins do also have a function in storing, detoxify and distributing metals throughout the cell ([[Team:Groningen/Literature#Merrifield2004|Merrifield 2004]], [[Team:Groningen/Literature#Gold2008|Gold 2008]]). These proteins have readily been used to create cell based systems for purification of contaminated water ([[Team:Groningen/Literature#Chen1998|Chen 1998]], [[Team:Groningen/Literature#Brady1994|Brady 1994]]). In addition to their wide application possibilities, they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one ([[Team:Groningen/Literature#Chang1998|Chang 1998]]).<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium ([[Team:Groningen/Literature#Deng2007|Deng 2007]]), arsenic ([[Team:Groningen/Literature#Ngu2006|Ngu 2006]], [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], [[Team:Groningen/Literature#Singh2008|Singh 2008]]), mercury ([[Team:Groningen/Literature#Chen1998|Chen 1998]], [[Team:Groningen/Literature#Chen1998|Chen 1997-2]], [[Team:Groningen/Literature#Deng2008|Deng 2008]]), nickel ([[Team:Groningen/Literature#Deng2003|Deng 2003]]) or a combination of metals ([[Team:Groningen/Literature#Chang1998|Chang 1998]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
Metal-protein complexes can be quantified using a fluorescent molecule ([[Team:Groningen/Literature#Cadosch2008 |Cadosch 2008]]) but Cu(I) binding to metallothioneins in metal thiolates, was shown to cause a concentration dependant increase in luminescence. These Cu(I) binding metallothioneins were shown to give rise to a Stokes shift of approximately 300nm upon excitation at 280nm ([[Team:Groningen/Literature#Beltramini1981|Beltramini 1981]], [[Team:Groningen/Literature#Gold2008|Gold 2008]]). <br />
<br />
<br />
<center>[[Image:800px-Zinc finger rendered.png|250px]] </center><br />
:Figure 1: Zinc finger protein, consisting of a α-helix and an anti-parallel β-sheet. The zinc atom (green) is bound by two histidines and two cysteins.<br />
<br />
=Cloning strategy=<br />
In order to have a functional accumulation device, the cDNA of a metallothionein (MT) will be amplified using [http://en.wikipedia.org/wiki/PCR PCR] and cloned into <partinfo>pSB1A2</partinfo>, also a corresponding metal-ion transporter was amplified by PCR and cloned behind the MT. Both will expressed by one promoter (constitutive or lactose inducible). In this way the bacterium will take up the metal-ion and consecutively the metal-ion will be sequestered by the MT. When this device is combined with the [[Team:Groningen/Project/Vesicle#Cloning_strategy|floating device]], the bacteria will start floating when a certain threshold of intracellular metal concentration is reached, because the negative regulator of the buoyancy device will be released and the gas vesicle cluster can be transcribed.<br />
<br />
[[Image:Accumulation device.PNG]]<br />
:Figure 2: Cloning strategy for the metal accumulation device. A promoter taken from <partinfo>J61002</partinfo> will be cloned in front of a metallothionein and a metal transporter in a <partinfo>pSB1A2</partinfo> vector. This device will be combined with the floating device.<br />
<br />
===Practical note===<br />
MTs are degraded intracellular inside lysozymes, especially when they are in the apo/non-bound state ([[Team:Groningen/Literature#Gold2008|Gold 2008]]), for bacteria the degradation rate is not known, but for <br />
mammalian MT this can be estimated around 0.8nmol apo-MT/mg protein/min ([[Team:Groningen/Literature#Klaassen1994|Klaassen 1994]]). This can be avoided by adding metal-salts (ZnCl, CuCl) to cells expressing the protein.<br />
<br />
=Metals=<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) ([[Team:Groningen/Literature#Ngu2006 |Ngu 2006]]) and fMT (the seaweed species ''Fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity ([[Team:Groningen/Literature#Singh2008 |Singh 2008]]), another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
===ArsR===<br />
ArsR is a trans-acting repressor that senses environmental As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and discriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified, its size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co-workers overexpressed ArsR in <i>E. coli</i> JM109 cells and found that the specific AS(III) content was 13-fold higher than the control without ArsR expression. High level expression of ArsR appeared to be toxic as a 3-fold reduction in cell density was observed. It has been shown that fusion partners reduce the toxicity of overexpression. Originally, Chen and co-workers made a fusion between ArsR and ELP (elastin protein), which is build out of VPGVG repeats. Because making a ArsR ELP153 fusion is very time consuming, we choose to make a fusion between MBP (maltose binding protein) and ArsR ([[Team:Groningen/Literature#Chen1998|Chen 1998]]).<br />
<BR><br />
Also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT was not successful, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
====Results====<br />
<br />
The fusion protein MBP-ArsR was built by creating giving the reverse primer of the MBP and the Forward primer of the ArsR a mutual restriction site SacI. The linker region was designed in such a way that it contained a Tev cleavage site, containing a SacI restriction site and a string of alanine residues to facilitate folding. The fusion protein has been succesfully cloned into the psb1AC3 vector creating biobrick {{part|BBa_K190027}}, but further attempts to add a promotor and rbs failed. Due to time the MBP-ArsR fusion protein has not been equipped with a promotor and so overexpression could not be established.<br />
<br />
===fMT===<br />
The <i>Fucus</i> [http://www.bioc.uzh.ch/mtpage/intro.html Metallothionein] (fMT) was isolated from the [http://en.wikipedia.org/wiki/Seaweed macroalgae] [http://en.wikipedia.org/wiki/Fucus_vesiculosus <i>Fucus vesiculosus</i> ]([[Team:Groningen/Literature#Morris1999|Morris 1999]]). It consists of 67 amino acid residues and has 16 cysteine residues, a high cysteine content is a key feature of MT. Another characteristic is the lack of aromatic residues is also seen in fMT where it only has one, tryptophan. Two domains containing cysteine residues are presumed to be involved in the metal binding function. Unusual in fMT is the presence of a 14 amino acid linker region between the two putative metal-binding domains which contains no cysteine residues. Plant MTs show this feature with about 40 residues, where vertebrate MTs only have three residues ([[Team:Groningen/Literature#Morris1999|Morris 1999]]). Being a MT fMT binds a multitude of metal ions, 6 Cd<sup>2+</sup> ions or 5 As<sup>3+</sup> ions in a sequential order, facilitated by the elongated linker domain {{todo|BUSY}}<br />
<br />
<br />
====Results====<br />
Arsenite uptake [[Team:Groningen/Protocols|assays]] were done to determine the As(III) accumulation of ''E. coli'' WT and fMT / GlpF overexpression strains. The concentration was measured by [[Team:Groningen/Protocols|ICP-MS]]. <br />
<br />
The arsenic uptake in ''E. coli'' WT (figure 3) as measured during this project (by [http://www.rikilt.wur.nl/NL/ RIKILT], Wageningen University), was compared with the uptake of ''E. coli'' with ArsR overexpression (described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], see figure 3). This shows that the arsenic uptake in ''E. coli'' WT behaves similar but has lower final As(III) uptake yield. The difference is about 10% in the standard mode, but a higher extracellular arsenic concentration seems to be needed to saturate the uptake of arsenic in ''E. coli'' WT compared to ''E. coli'' with ArsR overexpression. This can be seen by comparing the transition point to saturation in figure 3, which are respectively around 50µM As(III) and around 20µM. <br />
<br />
[[Image:As uptake in E coli ArsR overexp - Kostal 2004.PNG]]<br />
:Figure 3: Uptake of As(III) by ''E. coli'' WT (containing pSB1A2-pLac)<br />
<br />
There is a relatively large difference between the data generated by measuring the arsenic concentration with ICP-MS in the standard mode and measuring in the collusion cell technology mode (CCT mode). The difference between these two techniques is that in the standard mode it is possible that multi-atomic compounds lead to interference with the arsenic (mw = 75) peak, like argon-chloride (Ar = 40 + Cl = 35 (75%) or 37(25%)). Because 25% of this compound is found in the mw = 77 peak, a correction factor may be calculated to correct for this, the ICP-MS software (Thermo) automatically corrects for Ar-Cl interference. It uses the amount of Krypton and Selenium for this correction. In the CCT all multi-atomic compounds are supposed to be decomposed, therefore no interference will be found in this mode. But a disadvantage of this mode is that the resolution is 10x lower than the standard mode, leading to a smaller signal-to-noise ratio. Because of this, we decided to use the standard mode (corrected for interference) to determine the arsenic accumulation by ''E. coli''.<br />
<br />
A second arsenic measurement was performed (by [http://www.vwa.nl/portal/page?_pageid=119,1639634&_dad=portal&_schema=PORTAL Food and Consumer Product Safety Authority], Groningen) using ''E. coli'' WT and ''E. coli'' containing the [accumulation device] (<partinfo>BBa_K190038</partinfo>) and the different parts ([[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>)). The data was measured in the standard mode and the calculated arsenic imported by the cells is shown in figure 4.<br />
<br />
[[Image:As_uptake_in_WT_fMT_GlpF_ArsR.PNG]]<br />
:Figure 4: Uptake of As(III) by ''E. coli'' WT, and the strains containing the different parts of the accumulation device. As a control the arsenic uptake of ''E. coli'' with ArsR overexpression (as described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]]) is also shown.<br />
<br />
The curves in this figure show that there is no difference between the arsenic uptake by ''E. coli'' WT and by ''E. coli'' plus (parts of) the accumulation device. As a second observation, it can be seen that the uptake of arsenic in measured here is higher than found before (figure 3). A ratio of 2-3x was found for the WT strains (pSB1A2 and pArsR-RFP). These two differences will be discussed below. fMT shows exceptionally low arsenite uptake, this may be caused by incidentally "burning" the already dried cells at ~100;deg&C.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
====Discussion====<br />
Between the two data sets there are a few differences, first there seems to be no difference between arsenic uptake in WT and ''E. coli'' with the accumulation device (or parts of this). Secondly, the data of arsenic uptake by ''E. coli'' WT could was not reproducible and the last data set showed a arsenic uptake which was even higher for ''E. coli'' WT than the ''E. coli'' ArsR overexpression strain. <br />
<br />
*Why is there no difference between the ''E. coli'' WT and the ''E. coli'' with accumulation device?<br />
This can be caused by non-functional expression of one of the genes (fMT or GlpF) or both. For membrane proteins it is known that functional overexpression is harder than for cytoplasmic proteins ([[Team:Groningen/Literature#Lundstom2006|Lundstom 2006]]). This could be tested by doing As(III) uptake/binding experiments with purified proteins, but this requires protein purification which could be facilitated by the addition of a his-tag (not present yet). The function of the transporter can be tested by measuring the uptake in membrane vesicles and that of the accumulation protein can be tested by measuring metal binding for instance by isothermal titration calorimetry. Otherwise the proteins may not be produced at all, this should be tested by protein purification or sds-page. Another possibility is that these proteins cannot be produced by ‘’E. coli’’ at once, though functional expression was already proven by Singh ''et al.'' ([[Team:Groningen/Literature#Singh2008|Singh 2008]]).<br />
<br />
*Non reproducible concentrations of arsenic, imported by ''E. coli'' WT, which can be seen as there is a large difference (2-3x) in arsenic uptake determined from the first and the second measurement. All data from the second ICP-MS arsenic determination, were also unexpectedly higher than was found in literature ([[Team:Groningen/Literature#Kostal2004|Kostal 2004]], [[Team:Groningen/Literature#Singh2008|Singh 2008]]). This discrepancy may be caused by one of the following reasons.<br />
During the second arsenic uptake assay the time between the incubation and washing the cells was decreased to the minimum though during the first assay there was some time for the cells to export the As(III) via there exporter ArsB. This may have caused the lower uptake yield of arsenic in the first data set. Also there was a difference in cell concentration, in the second assay this was 2.5 times higher. It is presumably that with a higher cell concentration the uptake rate is slower but a saturating incubation time (>1hr) might cause that the equilibrium of arsenite concentration in/outside the cell is reached faster. After destruction of the samples of the first data set, the samples did not become a clear solution but a suspension containing white flakes. These were removed by centrifugation, but this seems to indicate incomplete destruction. This was not seen for the second samples, therefore an increased arsenite concentration may be measured as arsenite bound to the white flakes is not measured. <br />
It also might be, that during the second arsenite uptake assay the cells were washed less properly causing the concentration to become way higher than the first measurement. A more acidic buffer used for washing the cells is probably more efficient in removing metal ions than the TB74S buffer (pH 7.4), but as this protocol was the same as described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], this should be a major problem. The expected increase in arsenic concentration should be linear with the external arsenite concentration, but this was not seen (figure 4), a clear saturation curve was seen. <br />
A plausible cause is that there was a mistake in the calculations, a correction factor which was forgotten to correct for. Another plausible cause is that the concentration is higher because the measured concentration was for some samples 5 times higher than the calibration range. It might be that linear extrapolation is not correct. This can cause the structural increased arsenic uptake. <br />
<br />
*Other considerations:<br />
-Metal buffer interactions, causing a lower free-As(III) concentration surrounding the cell suspension.<br />
-Arsenic oxidation in aerobic conditions to As(V), this equilibrium may change over hours, so if the stock solution is enriched with As(V) it may take hours before it is changed to As(III) again. <br />
- Binding of other metal ions to the metallothionein causing competition for arsenite binding to fMT. Possible metal ions can be: Copper(I) or other metal ions present in the undefined LB medium. A requiry is that the metal ion should bind stronger or as strong to the MT as arsenite, which binds less strongly to MT than Zn(II) for instance or Cu(I).<br />
<br />
==Copper==<br />
<br />
===MymT===<br />
<br />
MymT is a 5kDa-protein which binds Cu(I) but also to less extend Zn(II) from ''Mycobaterterium tuberculosis''. This MT was found to bind 4-6 Cu(I) ions per molecule. Induction of the expression of MymT is the strongest with Cd and Cu. But upon over-expression of MymT in ''E. coli'', the protein becomes insoluble ([[Team:Groningen/Literature#Gold2008|Gold 2008]]). This may be caused by the fact that it is a protein from a gram-positive bacteria expressed in a gram negative bacterium. Therefore specialized cultivation conditions are needed, the cells should be grown at a low temperature (16 &deg;C). The functionality of MymT can be measured by fluorescence spectroscopy, as also found for other copper binding metallothioneins. Copper bound to MT create Cu-thiolates which can be excited at 280nm and gives a Stokes shift towards 600 nm ([[Team:Groningen/Literature#Beltramini1981|Beltramini 1981]]). <br />
<br />
'''Results:'''<br />
PCR on ''mym''T from pGB68 unfortunately did not give any correct cDNA fragments, even though the primer quality was improved ([[Team:Groningen/Protocols|Protocol Biobrick primers]]). Therefore the sub-project was discontinued.<br />
<br />
==Zinc==<br />
Below toxic concentrations, zinc is essential for many biological processes. Examples are enzymatic hydroxylation, DNA and RNA synthesis, transcription and translation, signal transduction and apoptosis regulation ([[http://en.wikipedia.org/wiki/Zinc 1]] and [[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]). Methallothioneins can adjust the zinc absorption up to 14-40%, though a real excess of zinc can be toxic. A daily intake of 100–300 mg Zn/day can give rise to copper / iron deficiency and damage of nerve receptors ([[Team:Groningen/Literature#Fosmire1990|Fosmire 1990]]). Examples of metallothioneins sequestering zinc, are SmtA from the cyanobacterium ''Synechococcus'' PCC7942 ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]), ZiaR from ''Synechocystis'' PCC 6803 ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]), human metallothioneins like MT-1 and -2. The mammalian proteins were found to bind 7 Zn<sup>2+</sup> ions by the thiolate-group of there cysteins.<br />
<br />
===SmtA===<br />
SmtA is a MT from ''Synechococcus'' PCC 6803, it was found to bind 3-4Zn ions and is supposed to have a function in preventing zinc toxicity ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]), but it also binds copper and cadmium ([[Team:Groningen/Literature#Shi1992|Shi 1992]]). Upon binding of Zn, the glutathione transferase fusion-protein showed a 1:3 stoichiometry and SmtA a 1:4 stoichiometry ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]). SmtA binds the 4 Zn ions via cystein thiolate-bridges, forming a Zn<sub>4</sub>Cys<sub>11</sub> cluster whichs was also found in mammalian MT, though these proteins do not have a homologous DNA sequence ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]). ''SmtA'' is found on an operon with it's transcriptional regulator ''smtB''. SmtB releases from the promoter-operator region in front of this operon, when it binds Zn via its metal binding motif. SmtB and [[Team:Groningen/Project/Accumulation#Arsenic|ArsR]] (negative transcriptional regulator binding arsenic) have similar functionalities but differ in metal binding motifs ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]). That ''Synechococcus'' is a gram negative bacterium might increase the possibility of functional and stable overexpression in ''E. coli'' .<br />
<br />
===Results===<br />
PCR reactions to amplify SmtA from pET29a and SmtA-GST from pGEX-3x were successful, but unfortunately the DNA sequence used to design the SmtA primers was not correct so therefore wrong cDNA fragments were amplified. Because it was too late to order new primers, this sub-project was discontinued.<br />
<br />
==Alternatives==<br />
{{todo|Inclusion bodies}} ([[Team:Groningen/Literature#Fowler1987|Fowler 1987]])<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
<!--==Inhibitory characteristics?==--><br />
<br />
==Modelling==<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
{{GraphHeader}}<br />
===Arsenic - ArsR===<br />
<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add up to a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal 2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Read inputs<br />
var awAs = getInput('awAs'); // g/mol<br />
var cAs = getInput('cAs') * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = getInput('Mcelldrywet'); // kg/kg<br />
var rhocell = getInput('rhocell'); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set outputs<br />
setOutput('Aspercellvolume', Aspercellvolume);<br />
setOutput('molAspercellvolume', molAspercellvolume);<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|frame]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right, a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
In addition to binding to As(III), ArsR can repress Ars, creating a negative feedback loop. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen 1997]]). In the <i>E. coli</i> top 10 there is only ars promoter present on the genome to produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD, but these are not used in this project. We intend to introduce instead a constitutive promoter (pro), which produces just ArsR, in order to bind as much As(III) as possible.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary.<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
KR<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
<nobr>KA<sub>d</sub> (ArsR<sub>ars</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)</nobr><br/><br />
KM<sub>d</sub> (MBPArsR<sub>As</sub>) = <input type="text" id="KMd" value="6"/> &micro;M (???)<br/><br />
KF<sub>d</sub> (fMTArsR<sub>As</sub>) = <input type="text" id="KFd" value="6"/> &micro;M (???)<br/><br />
n<sub>f</sub> = <input type="text" id="nf" value="3"/> (???)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;R (ArsR) = <input type="text" id="tauR" value="0.1"/> min (???)<br/><br />
&tau;M (MBPArsR) = <input type="text" id="tauM" value="0.1"/> min (???)<br/><br />
&tau;F (fMT) = <input type="text" id="tauF" value="0.1"/> min (???)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
<nobr>&beta;RN (ars1 &rarr; ArsR) = <input type="text" id="beta1" value="100"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;R (proR &rarr; ArsR) = <input type="text" id="beta3" value="100"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;M (proM &rarr; MBPArsR) = <input type="text" id="betaM" value="26.6"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;F (proF &rarr; fMT) = <input type="text" id="betaF" value="200"/> 1/second (???)</nobr><br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
ars1<sub>total</sub> = <input type="text" id="ars1TPerCell" value="1"/> per cell<br/><br />
<nobr>proR = <input type="text" id="proRPerCell" value="0"/> per cell (??)</nobr><br/><br />
<nobr>proM = <input type="text" id="proMPerCell" value="100"/> per cell (??)</nobr><br/><br />
<nobr>proF = <input type="text" id="proFPerCell" value="0"/> per cell (??)</nobr><br/><br />
V<sub>cell</sub> = <input type="text" id="Vcell" value="1"/> &micro;m<sup>3</sup> </html><br />
([http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| CCBD])<html><br />
<br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>ArsR</dt><br />
<dd><br />
ars / ars<sub>total</sub> = <span id="arsFraction"></span><br/><br />
ArsR = <span id="ArsR"></span> &micro;M<br/><br />
<!--ArsR<sub>total</sub> = <span id="ArsRT"></span> &micro;M<br/>--><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="AsinT"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="AsinTfactor"></span><br/><br />
</dd><br />
</dl><br />
</html><br />
<span id="accumulationFactorData"></span><br />
{{graph|Team:Groningen/Graphs/AccumulationFactor|id=accumulationFactorGraph}}<br />
(For constants other than the ones on the left the [[Team:Groningen/Modelling/Arsenic.js|default values]] are used.)<br />
<html><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Intermediates (mostly useful for debugging)<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Read inputs<br />
var c = arsenicModelConstants();<br />
c.AsT = 0;<br />
c.K1d = getInput('K1d') * 1e-6; // micromolar -> molar<br />
c.K3d2 = Math.pow(getInput('K3d') * 1e-6,2); // micromolar -> molar<br />
c.KMd = getInput('KMd') * 1e-6; // micromolar -> molar<br />
c.KFd = getInput('KFd') * 1e-6; // micromolar -> molar<br />
c.nf = getInput('nf') * 1e-6; // micromolar -> molar<br />
c.tauR = getInput('tauR') * 60; // minutes -> seconds<br />
c.tauM = getInput('tauM') * 60; // minutes -> seconds<br />
c.tauF = getInput('tauF') * 60; // minutes -> seconds<br />
c.beta1 = getInput('beta1'); // 1/second<br />
c.beta3 = getInput('beta3'); // 1/second<br />
c.betaM = getInput('betaM'); // 1/second<br />
c.betaF = getInput('betaF'); // 1/second<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var Vcell = getInput('Vcell') * 1e-15; // micrometer^3/cell -> liter/cell<br />
c.ars1T = getInput('ars1TPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.ars2T = 0;<br />
c.pro = getInput('proRPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.proM = getInput('proMPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.proF = getInput('proFPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var x = arsenicModelEquilibrium(c);<br />
var ArsR = x._ArsR;<br />
var arsFraction = x._arsF;<br />
var AsinTfactor = 1 + ArsR/c.K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
setOutput('arsFraction', arsFraction);<br />
setOutput('ArsR', ArsR * 1e6);<br />
<br />
// Set outputs<br />
setOutput('AsinTfactor', AsinTfactor);<br />
<br />
// Draw graph<br />
var dataNode = document.getElementById("accumulationFactorData");<br />
var graphNode = document.getElementById("accumulationFactorGraph");<br />
var data = {AsT:[], AsexT0:[], AsinT:[], AsinT2:[]};<br />
var bAsin, cAsin, Asin;<br />
// Some guesses<br />
var c2 = {}, x2;<br />
for(var a in c) c2[a] = c[a];<br />
c2.v5 *= 2;<br />
for(var AsexT0uM=0.1; AsexT0uM<=100; AsexT0uM*=1.1) {<br />
c.AsT = c.Vs*AsexT0uM*1e-6;<br />
c2.AsT = c.AsT;<br />
x = arsenicModelEquilibrium(c);<br />
x2 = arsenicModelEquilibrium(c2);<br />
data.AsT.push(c.AsT);<br />
data.AsexT0.push(c.AsT/c.Vs);<br />
data.AsinT.push(x.AsinT*c.Vc/c.AsT);<br />
data.AsinT2.push(x2.AsinT*c.Vc/c.AsT);<br />
}<br />
dataNode.data = data;<br />
if (graphNode.refresh) graphNode.refresh();<br />
}<br />
</script><br />
</html><br />
<br />
'''In conclusion:'''<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it should not matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for Ars promotors, you can see that it is clearly advantageous to use constitutive promotors as just adding ars promoters does not increase the accumulation factor. A plasmid containing an ars promoter and (just) a gene coding for ArsR behind it might contain more arsenic, but there would also be more unbound arsenic, increasing the toxicity.<br />
* The model is not very sensitive to different values for K3d (with K3d=1mM the accumulation factor is 248.06 and with K3d=1nM it is 240.57).<br />
* The accumulation factor is greatly affected by the product of the half-life of ArsR and the production rate.<br />
<br />
<!-- ==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies [[Team:Groningen/Literature#Lin2007-2|Lin 2007]]<br />
**** Inductively coupled mass spectrometry (ICP-MS) ([[Team:Groningen/Literature#Meng2004|Meng 2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen 1997]]). In the paper ArsD is purified, but if that is not feasible for us, we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest--><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-10-21T12:52:47Z<p>Frans: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
<div class="introduction"><br />
=Accumulation=<br />
Once heavy metals have entered the cell, it is crucial to keep them there. As these metals are toxic to cell survival in critical amounts, evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variety of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..</div><br />
<br />
=Metallothioneins=<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). The contain a conserved cys-x-cys or cys-x-his motif which coordinates metal binding, as can be seen in figure 1. They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity (Kb), they are ''in vivo'' used as a defense against oxidative stress by chelating metals. This proteins do also have a function in storing, detoxify and distributing metals throughout the cell ([[Team:Groningen/Literature#Merrifield2004|Merrifield 2004]], [[Team:Groningen/Literature#Gold2008|Gold 2008]]). These proteins have readily been used to create cell based systems for purification of contaminated water ([[Team:Groningen/Literature#Chen1998|Chen 1998]], [[Team:Groningen/Literature#Brady1994|Brady 1994]]). In addition to their wide application possibilities, they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one ([[Team:Groningen/Literature#Chang1998|Chang 1998]]).<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium ([[Team:Groningen/Literature#Deng2007|Deng 2007]]), arsenic ([[Team:Groningen/Literature#Ngu2006|Ngu 2006]], [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], [[Team:Groningen/Literature#Singh2008|Singh 2008]]), mercury ([[Team:Groningen/Literature#Chen1998|Chen 1998]], [[Team:Groningen/Literature#Chen1998|Chen 1997-2]], [[Team:Groningen/Literature#Deng2008|Deng 2008]]), nickel ([[Team:Groningen/Literature#Deng2003|Deng 2003]]) or a combination of metals ([[Team:Groningen/Literature#Chang1998|Chang 1998]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
Metal-protein complexes can be quantified using a fluorescent molecule ([[Team:Groningen/Literature#Cadosch2008 |Cadosch 2008]]) but Cu(I) binding to metallothioneins in metal thiolates, was shown to cause a concentration dependant increase in luminescence. These Cu(I) binding metallothioneins were shown to give rise to a Stokes shift of approximately 300nm upon excitation at 280nm ([[Team:Groningen/Literature#Beltramini1981|Beltramini 1981]], [[Team:Groningen/Literature#Gold2008|Gold 2008]]). <br />
<br />
<br />
<center>[[Image:800px-Zinc finger rendered.png|250px]] </center><br />
:Figure 1: Zinc finger protein, consisting of a α-helix and an anti-parallel β-sheet. The zinc atom (green) is bound by two histidines and two cysteins.<br />
<br />
=Cloning strategy=<br />
In order to have a functional accumulation device, the cDNA of a metallothionein (MT) will be amplified using [http://en.wikipedia.org/wiki/PCR PCR] and cloned into <partinfo>pSB1A2</partinfo>, also a corresponding metal-ion transporter was amplified by PCR and cloned behind the MT. Both will expressed by one promoter (constitutive or lactose inducible). In this way the bacterium will take up the metal-ion and consecutively the metal-ion will be sequestered by the MT. When this device is combined with the [[Team:Groningen/Project/Vesicle#Cloning_strategy|floating device]], the bacteria will start floating when a certain threshold of intracellular metal concentration is reached, because the negative regulator of the buoyancy device will be released and the gas vesicle cluster can be transcribed.<br />
<br />
[[Image:Accumulation device.PNG]]<br />
:Figure 2: Cloning strategy for the metal accumulation device. A promoter taken from <partinfo>J61002</partinfo> will be cloned in front of a metallothionein and a metal transporter in a <partinfo>pSB1A2</partinfo> vector. This device will be combined with the floating device.<br />
<br />
===Practical note===<br />
MTs are degraded intracellular inside lysozymes, especially when they are in the apo/non-bound state ([[Team:Groningen/Literature#Gold2008|Gold 2008]]), for bacteria the degradation rate is not known, but for <br />
mammalian MT this can be estimated around 0.8nmol apo-MT/mg protein/min ([[Team:Groningen/Literature#Klaassen1994|Klaassen 1994]]). This can be avoided by adding metal-salts (ZnCl, CuCl) to cells expressing the protein.<br />
<br />
=Metals=<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) ([[Team:Groningen/Literature#Ngu2006 |Ngu 2006]]) and fMT (the seaweed species ''Fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity ([[Team:Groningen/Literature#Singh2008 |Singh 2008]]), another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
===ArsR===<br />
ArsR is a trans-acting repressor that senses environmental As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and discriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified, its size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co-workers overexpressed ArsR in <i>E. coli</i> JM109 cells and found that the specific AS(III) content was 13-fold higher than the control without ArsR expression. High level expression of ArsR appeared to be toxic as a 3-fold reduction in cell density was observed. It has been shown that fusion partners reduce the toxicity of overexpression. Originally, Chen and co-workers made a fusion between ArsR and ELP (elastin protein), which is build out of VPGVG repeats. Because making a ArsR ELP153 fusion is very time consuming, we choose to make a fusion between MBP (maltose binding protein) and ArsR ([[Team:Groningen/Literature#Chen1998|Chen 1998]]).<br />
<BR><br />
Also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT was not successful, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
====Results====<br />
<br />
The fusion protein MBP-ArsR was built by creating giving the reverse primer of the MBP and the Forward primer of the ArsR a mutual restriction site SacI. The linker region was designed in such a way that it contained a Tev cleavage site, containing a SacI restriction site and a string of alanine residues to facilitate folding. The fusion protein has been sucesfully cloned into the psb1AC3 vector, but further attempts to add a promotor and rbs failed. Due to time the MBP-ArsR fusion protein has not been equipped with a promotor and so overexpression could not be established.<br />
<br />
===fMT===<br />
The <i>Fucus</i> [http://www.bioc.uzh.ch/mtpage/intro.html Metallothionein] (fMT) was isolated from the [http://en.wikipedia.org/wiki/Seaweed macroalgae] [http://en.wikipedia.org/wiki/Fucus_vesiculosus <i>Fucus vesiculosus</i> ]([[Team:Groningen/Literature#Morris1999|Morris 1999]]). It consists of 67 amino acid residues and has 16 cysteine residues, a high cysteine content is a key feature of MT. Another characteristic is the lack of aromatic residues is also seen in fMT where it only has one, tryptophan. Two domains containing cysteine residues are presumed to be involved in the metal binding function. Unusual in fMT is the presence of a 14 amino acid linker region between the two putative metal-binding domains which contains no cysteine residues. Plant MTs show this feature with about 40 residues, where vertebrate MTs only have three residues ([[Team:Groningen/Literature#Morris1999|Morris 1999]]). Being a MT fMT binds a multitude of metal ions, 6 Cd<sup>2+</sup> ions or 5 As<sup>3+</sup> ions in a sequential order, facilitated by the elongated linker domain {{todo|BUSY}}<br />
<br />
<br />
====Results====<br />
Arsenite uptake [[Team:Groningen/Protocols|assays]] were done to determine the As(III) accumulation of ''E. coli'' WT and fMT / GlpF overexpression strains. The concentration was measured by [[Team:Groningen/Protocols|ICP-MS]]. <br />
<br />
The arsenic uptake in ''E. coli'' WT (figure 3) as measured during this project (by [http://www.rikilt.wur.nl/NL/ RIKILT], Wageningen University), was compared with the uptake of ''E. coli'' with ArsR overexpression (described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], see figure 3). This shows that the arsenic uptake in ''E. coli'' WT behaves similar but has lower final As(III) uptake yield. The difference is about 10% in the standard mode, but a higher extracellular arsenic concentration seems to be needed to saturate the uptake of arsenic in ''E. coli'' WT compared to ''E. coli'' with ArsR overexpression. This can be seen by comparing the transition point to saturation in figure 3, which are respectively around 50µM As(III) and around 20µM. <br />
<br />
[[Image:As uptake in E coli ArsR overexp - Kostal 2004.PNG]]<br />
:Figure 3: Uptake of As(III) by ''E. coli'' WT (containing pSB1A2-pLac)<br />
<br />
There is a relatively large difference between the data generated by measuring the arsenic concentration with ICP-MS in the standard mode and measuring in the collusion cell technology mode (CCT mode). The difference between these two techniques is that in the standard mode it is possible that multi-atomic compounds lead to interference with the arsenic (mw = 75) peak, like argon-chloride (Ar = 40 + Cl = 35 (75%) or 37(25%)). Because 25% of this compound is found in the mw = 77 peak, a correction factor may be calculated to correct for this, the ICP-MS software (Thermo) automatically corrects for Ar-Cl interference. It uses the amount of Krypton and Selenium for this correction. In the CCT all multi-atomic compounds are supposed to be decomposed, therefore no interference will be found in this mode. But a disadvantage of this mode is that the resolution is 10x lower than the standard mode, leading to a smaller signal-to-noise ratio. Because of this, we decided to use the standard mode (corrected for interference) to determine the arsenic accumulation by ''E. coli''.<br />
<br />
A second arsenic measurement was performed (by [http://www.vwa.nl/portal/page?_pageid=119,1639634&_dad=portal&_schema=PORTAL Food and Consumer Product Safety Authority], Groningen) using ''E. coli'' WT and ''E. coli'' containing the [accumulation device] (<partinfo>BBa_K190038</partinfo>) and the different parts ([[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>)). The data was measured in the standard mode and the calculated arsenic imported by the cells is shown in figure 4.<br />
<br />
[[Image:As_uptake_in_WT_fMT_GlpF_ArsR.PNG]]<br />
:Figure 4: Uptake of As(III) by ''E. coli'' WT, and the strains containing the different parts of the accumulation device. As a control the arsenic uptake of ''E. coli'' with ArsR overexpression (as described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]]) is also shown.<br />
<br />
The curves in this figure show that there is no difference between the arsenic uptake by ''E. coli'' WT and by ''E. coli'' plus (parts of) the accumulation device. As a second observation, it can be seen that the uptake of arsenic in measured here is higher than found before (figure 3). A ratio of 2-3x was found for the WT strains (pSB1A2 and pArsR-RFP). These two differences will be discussed below. fMT shows exceptionally low arsenite uptake, this may be caused by incidentally "burning" the already dried cells at ~100;deg&C.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
====Discussion====<br />
Between the two data sets there are a few differences, first there seems to be no difference between arsenic uptake in WT and ''E. coli'' with the accumulation device (or parts of this). Secondly, the data of arsenic uptake by ''E. coli'' WT could was not reproducible and the last data set showed a arsenic uptake which was even higher for ''E. coli'' WT than the ''E. coli'' ArsR overexpression strain. <br />
<br />
*Why is there no difference between the ''E. coli'' WT and the ''E. coli'' with accumulation device?<br />
This can be caused by non-functional expression of one of the genes (fMT or GlpF) or both. For membrane proteins it is known that functional overexpression is harder than for cytoplasmic proteins ([[Team:Groningen/Literature#Lundstom2006|Lundstom 2006]]). This could be tested by doing As(III) uptake/binding experiments with purified proteins, but this requires protein purification which could be facilitated by the addition of a his-tag (not present yet). The function of the transporter can be tested by measuring the uptake in membrane vesicles and that of the accumulation protein can be tested by measuring metal binding for instance by isothermal titration calorimetry. Otherwise the proteins may not be produced at all, this should be tested by protein purification or sds-page. Another possibility is that these proteins cannot be produced by ‘’E. coli’’ at once, though functional expression was already proven by Singh ''et al.'' ([[Team:Groningen/Literature#Singh2008|Singh 2008]]).<br />
<br />
*Non reproducible concentrations of arsenic, imported by ''E. coli'' WT, which can be seen as there is a large difference (2-3x) in arsenic uptake determined from the first and the second measurement. All data from the second ICP-MS arsenic determination, were also unexpectedly higher than was found in literature ([[Team:Groningen/Literature#Kostal2004|Kostal 2004]], [[Team:Groningen/Literature#Singh2008|Singh 2008]]). This discrepancy may be caused by one of the following reasons.<br />
During the second arsenic uptake assay the time between the incubation and washing the cells was decreased to the minimum though during the first assay there was some time for the cells to export the As(III) via there exporter ArsB. This may have caused the lower uptake yield of arsenic in the first data set. Also there was a difference in cell concentration, in the second assay this was 2.5 times higher. It is presumably that with a higher cell concentration the uptake rate is slower but a saturating incubation time (>1hr) might cause that the equilibrium of arsenite concentration in/outside the cell is reached faster. After destruction of the samples of the first data set, the samples did not become a clear solution but a suspension containing white flakes. These were removed by centrifugation, but this seems to indicate incomplete destruction. This was not seen for the second samples, therefore an increased arsenite concentration may be measured as arsenite bound to the white flakes is not measured. <br />
It also might be, that during the second arsenite uptake assay the cells were washed less properly causing the concentration to become way higher than the first measurement. A more acidic buffer used for washing the cells is probably more efficient in removing metal ions than the TB74S buffer (pH 7.4), but as this protocol was the same as described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], this should be a major problem. The expected increase in arsenic concentration should be linear with the external arsenite concentration, but this was not seen (figure 4), a clear saturation curve was seen. <br />
A plausible cause is that there was a mistake in the calculations, a correction factor which was forgotten to correct for. Another plausible cause is that the concentration is higher because the measured concentration was for some samples 5 times higher than the calibration range. It might be that linear extrapolation is not correct. This can cause the structural increased arsenic uptake. <br />
<br />
*Other considerations:<br />
-Metal buffer interactions, causing a lower free-As(III) concentration surrounding the cell suspension.<br />
-Arsenic oxidation in aerobic conditions to As(V), this equilibrium may change over hours, so if the stock solution is enriched with As(V) it may take hours before it is changed to As(III) again. <br />
- Binding of other metal ions to the metallothionein causing competition for arsenite binding to fMT. Possible metal ions can be: Copper(I) or other metal ions present in the undefined LB medium. A requiry is that the metal ion should bind stronger or as strong to the MT as arsenite, which binds less strongly to MT than Zn(II) for instance or Cu(I).<br />
<br />
==Copper==<br />
<br />
===MymT===<br />
<br />
MymT is a 5kDa-protein which binds Cu(I) but also to less extend Zn(II) from ''Mycobaterterium tuberculosis''. This MT was found to bind 4-6 Cu(I) ions per molecule. Induction of the expression of MymT is the strongest with Cd and Cu. But upon over-expression of MymT in ''E. coli'', the protein becomes insoluble ([[Team:Groningen/Literature#Gold2008|Gold 2008]]). This may be caused by the fact that it is a protein from a gram-positive bacteria expressed in a gram negative bacterium. Therefore specialized cultivation conditions are needed, the cells should be grown at a low temperature (16 &deg;C). The functionality of MymT can be measured by fluorescence spectroscopy, as also found for other copper binding metallothioneins. Copper bound to MT create Cu-thiolates which can be excited at 280nm and gives a Stokes shift towards 600 nm ([[Team:Groningen/Literature#Beltramini1981|Beltramini 1981]]). <br />
<br />
'''Results:'''<br />
PCR on ''mym''T from pGB68 unfortunately did not give any correct cDNA fragments, even though the primer quality was improved ([[Team:Groningen/Protocols|Protocol Biobrick primers]]). Therefore the sub-project was discontinued.<br />
<br />
==Zinc==<br />
Below toxic concentrations, zinc is essential for many biological processes. Examples are enzymatic hydroxylation, DNA and RNA synthesis, transcription and translation, signal transduction and apoptosis regulation ([[http://en.wikipedia.org/wiki/Zinc 1]] and [[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]). Methallothioneins can adjust the zinc absorption up to 14-40%, though a real excess of zinc can be toxic. A daily intake of 100–300 mg Zn/day can give rise to copper / iron deficiency and damage of nerve receptors ([[Team:Groningen/Literature#Fosmire1990|Fosmire 1990]]). Examples of metallothioneins sequestering zinc, are SmtA from the cyanobacterium ''Synechococcus'' PCC7942 ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]), ZiaR from ''Synechocystis'' PCC 6803 ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]), human metallothioneins like MT-1 and -2. The mammalian proteins were found to bind 7 Zn<sup>2+</sup> ions by the thiolate-group of there cysteins.<br />
<br />
===SmtA===<br />
SmtA is a MT from ''Synechococcus'' PCC 6803, it was found to bind 3-4Zn ions and is supposed to have a function in preventing zinc toxicity ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]), but it also binds copper and cadmium ([[Team:Groningen/Literature#Shi1992|Shi 1992]]). Upon binding of Zn, the glutathione transferase fusion-protein showed a 1:3 stoichiometry and SmtA a 1:4 stoichiometry ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]). SmtA binds the 4 Zn ions via cystein thiolate-bridges, forming a Zn<sub>4</sub>Cys<sub>11</sub> cluster whichs was also found in mammalian MT, though these proteins do not have a homologous DNA sequence ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]). ''SmtA'' is found on an operon with it's transcriptional regulator ''smtB''. SmtB releases from the promoter-operator region in front of this operon, when it binds Zn via its metal binding motif. SmtB and [[Team:Groningen/Project/Accumulation#Arsenic|ArsR]] (negative transcriptional regulator binding arsenic) have similar functionalities but differ in metal binding motifs ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]). That ''Synechococcus'' is a gram negative bacterium might increase the possibility of functional and stable overexpression in ''E. coli'' .<br />
<br />
===Results===<br />
PCR reactions to amplify SmtA from pET29a and SmtA-GST from pGEX-3x were successful, but unfortunately the DNA sequence used to design the SmtA primers was not correct so therefore wrong cDNA fragments were amplified. Because it was too late to order new primers, this sub-project was discontinued.<br />
<br />
==Alternatives==<br />
{{todo|Inclusion bodies}} ([[Team:Groningen/Literature#Fowler1987|Fowler 1987]])<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
<!--==Inhibitory characteristics?==--><br />
<br />
==Modelling==<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
{{GraphHeader}}<br />
===Arsenic - ArsR===<br />
<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add up to a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal 2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Read inputs<br />
var awAs = getInput('awAs'); // g/mol<br />
var cAs = getInput('cAs') * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = getInput('Mcelldrywet'); // kg/kg<br />
var rhocell = getInput('rhocell'); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set outputs<br />
setOutput('Aspercellvolume', Aspercellvolume);<br />
setOutput('molAspercellvolume', molAspercellvolume);<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|frame]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right, a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
In addition to binding to As(III), ArsR can repress Ars, creating a negative feedback loop. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen 1997]]). In the <i>E. coli</i> top 10 there is only ars promoter present on the genome to produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD, but these are not used in this project. We intend to introduce instead a constitutive promoter (pro), which produces just ArsR, in order to bind as much As(III) as possible.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary.<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
KR<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
<nobr>KA<sub>d</sub> (ArsR<sub>ars</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)</nobr><br/><br />
KM<sub>d</sub> (MBPArsR<sub>As</sub>) = <input type="text" id="KMd" value="6"/> &micro;M (???)<br/><br />
KF<sub>d</sub> (fMTArsR<sub>As</sub>) = <input type="text" id="KFd" value="6"/> &micro;M (???)<br/><br />
n<sub>f</sub> = <input type="text" id="nf" value="3"/> (???)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;R (ArsR) = <input type="text" id="tauR" value="0.1"/> min (???)<br/><br />
&tau;M (MBPArsR) = <input type="text" id="tauM" value="0.1"/> min (???)<br/><br />
&tau;F (fMT) = <input type="text" id="tauF" value="0.1"/> min (???)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
<nobr>&beta;RN (ars1 &rarr; ArsR) = <input type="text" id="beta1" value="100"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;R (proR &rarr; ArsR) = <input type="text" id="beta3" value="100"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;M (proM &rarr; MBPArsR) = <input type="text" id="betaM" value="26.6"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;F (proF &rarr; fMT) = <input type="text" id="betaF" value="200"/> 1/second (???)</nobr><br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
ars1<sub>total</sub> = <input type="text" id="ars1TPerCell" value="1"/> per cell<br/><br />
<nobr>proR = <input type="text" id="proRPerCell" value="0"/> per cell (??)</nobr><br/><br />
<nobr>proM = <input type="text" id="proMPerCell" value="100"/> per cell (??)</nobr><br/><br />
<nobr>proF = <input type="text" id="proFPerCell" value="0"/> per cell (??)</nobr><br/><br />
V<sub>cell</sub> = <input type="text" id="Vcell" value="1"/> &micro;m<sup>3</sup> </html><br />
([http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| CCBD])<html><br />
<br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>ArsR</dt><br />
<dd><br />
ars / ars<sub>total</sub> = <span id="arsFraction"></span><br/><br />
ArsR = <span id="ArsR"></span> &micro;M<br/><br />
<!--ArsR<sub>total</sub> = <span id="ArsRT"></span> &micro;M<br/>--><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="AsinT"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="AsinTfactor"></span><br/><br />
</dd><br />
</dl><br />
</html><br />
<span id="accumulationFactorData"></span><br />
{{graph|Team:Groningen/Graphs/AccumulationFactor|id=accumulationFactorGraph}}<br />
(For constants other than the ones on the left the [[Team:Groningen/Modelling/Arsenic.js|default values]] are used.)<br />
<html><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Intermediates (mostly useful for debugging)<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Read inputs<br />
var c = arsenicModelConstants();<br />
c.AsT = 0;<br />
c.K1d = getInput('K1d') * 1e-6; // micromolar -> molar<br />
c.K3d2 = Math.pow(getInput('K3d') * 1e-6,2); // micromolar -> molar<br />
c.KMd = getInput('KMd') * 1e-6; // micromolar -> molar<br />
c.KFd = getInput('KFd') * 1e-6; // micromolar -> molar<br />
c.nf = getInput('nf') * 1e-6; // micromolar -> molar<br />
c.tauR = getInput('tauR') * 60; // minutes -> seconds<br />
c.tauM = getInput('tauM') * 60; // minutes -> seconds<br />
c.tauF = getInput('tauF') * 60; // minutes -> seconds<br />
c.beta1 = getInput('beta1'); // 1/second<br />
c.beta3 = getInput('beta3'); // 1/second<br />
c.betaM = getInput('betaM'); // 1/second<br />
c.betaF = getInput('betaF'); // 1/second<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var Vcell = getInput('Vcell') * 1e-15; // micrometer^3/cell -> liter/cell<br />
c.ars1T = getInput('ars1TPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.ars2T = 0;<br />
c.pro = getInput('proRPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.proM = getInput('proMPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.proF = getInput('proFPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var x = arsenicModelEquilibrium(c);<br />
var ArsR = x._ArsR;<br />
var arsFraction = x._arsF;<br />
var AsinTfactor = 1 + ArsR/c.K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
setOutput('arsFraction', arsFraction);<br />
setOutput('ArsR', ArsR * 1e6);<br />
<br />
// Set outputs<br />
setOutput('AsinTfactor', AsinTfactor);<br />
<br />
// Draw graph<br />
var dataNode = document.getElementById("accumulationFactorData");<br />
var graphNode = document.getElementById("accumulationFactorGraph");<br />
var data = {AsT:[], AsexT0:[], AsinT:[], AsinT2:[]};<br />
var bAsin, cAsin, Asin;<br />
// Some guesses<br />
var c2 = {}, x2;<br />
for(var a in c) c2[a] = c[a];<br />
c2.v5 *= 2;<br />
for(var AsexT0uM=0.1; AsexT0uM<=100; AsexT0uM*=1.1) {<br />
c.AsT = c.Vs*AsexT0uM*1e-6;<br />
c2.AsT = c.AsT;<br />
x = arsenicModelEquilibrium(c);<br />
x2 = arsenicModelEquilibrium(c2);<br />
data.AsT.push(c.AsT);<br />
data.AsexT0.push(c.AsT/c.Vs);<br />
data.AsinT.push(x.AsinT*c.Vc/c.AsT);<br />
data.AsinT2.push(x2.AsinT*c.Vc/c.AsT);<br />
}<br />
dataNode.data = data;<br />
if (graphNode.refresh) graphNode.refresh();<br />
}<br />
</script><br />
</html><br />
<br />
'''In conclusion:'''<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it should not matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for Ars promotors, you can see that it is clearly advantageous to use constitutive promotors as just adding ars promoters does not increase the accumulation factor. A plasmid containing an ars promoter and (just) a gene coding for ArsR behind it might contain more arsenic, but there would also be more unbound arsenic, increasing the toxicity.<br />
* The model is not very sensitive to different values for K3d (with K3d=1mM the accumulation factor is 248.06 and with K3d=1nM it is 240.57).<br />
* The accumulation factor is greatly affected by the product of the half-life of ArsR and the production rate.<br />
<br />
<!-- ==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies [[Team:Groningen/Literature#Lin2007-2|Lin 2007]]<br />
**** Inductively coupled mass spectrometry (ICP-MS) ([[Team:Groningen/Literature#Meng2004|Meng 2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen 1997]]). In the paper ArsD is purified, but if that is not feasible for us, we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest--><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-10-21T12:24:59Z<p>Frans: /* ArsR */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
<div class="introduction"><br />
=Accumulation=<br />
Once heavy metals have entered the cell, it is crucial to keep them there. As these metals are toxic to cell survival in critical amounts, evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variety of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..</div><br />
<br />
=Metallothioneins=<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). The contain a conserved cys-x-cys or cys-x-his motif which coordinates metal binding, as can be seen in figure 1. They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity (Kb), they are ''in vivo'' used as a defense against oxidative stress by chelating metals. This proteins do also have a function in storing, detoxify and distributing metals throughout the cell ([[Team:Groningen/Literature#Merrifield2004|Merrifield 2004]], [[Team:Groningen/Literature#Gold2008|Gold 2008]]). These proteins have readily been used to create cell based systems for purification of contaminated water ([[Team:Groningen/Literature#Chen1998|Chen 1998]], [[Team:Groningen/Literature#Brady1994|Brady 1994]]). In addition to their wide application possibilities, they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one ([[Team:Groningen/Literature#Chang1998|Chang 1998]]).<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium ([[Team:Groningen/Literature#Deng2007|Deng 2007]]), arsenic ([[Team:Groningen/Literature#Ngu2006|Ngu 2006]], [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], [[Team:Groningen/Literature#Singh2008|Singh 2008]]), mercury ([[Team:Groningen/Literature#Chen1998|Chen 1998]], [[Team:Groningen/Literature#Chen1998|Chen 1997-2]], [[Team:Groningen/Literature#Deng2008|Deng 2008]]), nickel ([[Team:Groningen/Literature#Deng2003|Deng 2003]]) or a combination of metals ([[Team:Groningen/Literature#Chang1998|Chang 1998]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
Metal-protein complexes can be quantified using a fluorescent molecule ([[Team:Groningen/Literature#Cadosch2008 |Cadosch 2008]]) but Cu(I) binding to metallothioneins in metal thiolates, was shown to cause a concentration dependant increase in luminescence. These Cu(I) binding metallothioneins were shown to give rise to a Stokes shift of approximately 300nm upon excitation at 280nm ([[Team:Groningen/Literature#Beltramini1981|Beltramini 1981]], [[Team:Groningen/Literature#Gold2008|Gold 2008]]). <br />
<br />
<br />
<center>[[Image:800px-Zinc finger rendered.png|250px]] </center><br />
:Figure 1: Zinc finger protein, consisting of a α-helix and an anti-parallel β-sheet. The zinc atom (green) is bound by two histidines and two cysteins.<br />
<br />
=Cloning strategy=<br />
In order to have a functional accumulation device, the cDNA of a metallothionein (MT) will be amplified using [http://en.wikipedia.org/wiki/PCR PCR] and cloned into <partinfo>pSB1A2</partinfo>, also a corresponding metal-ion transporter was amplified by PCR and cloned behind the MT. Both will expressed by one promoter (constitutive or lactose inducible). In this way the bacterium will take up the metal-ion and consecutively the metal-ion will be sequestered by the MT. When this device is combined with the [[Team:Groningen/Project/Vesicle#Cloning_strategy|floating device]], the bacteria will start floating when a certain threshold of intracellular metal concentration is reached, because the negative regulator of the buoyancy device will be released and the gas vesicle cluster can be transcribed.<br />
<br />
[[Image:Accumulation device.PNG]]<br />
:Figure 2: Cloning strategy for the metal accumulation device. A promoter taken from <partinfo>J61002</partinfo> will be cloned in front of a metallothionein and a metal transporter in a <partinfo>pSB1A2</partinfo> vector. This device will be combined with the floating device.<br />
<br />
===Practical note===<br />
MTs are degraded intracellular inside lysozymes, especially when they are in the apo/non-bound state ([[Team:Groningen/Literature#Gold2008|Gold 2008]]), for bacteria the degradation rate is not known, but for <br />
mammalian MT this can be estimated around 0.8nmol apo-MT/mg protein/min ([[Team:Groningen/Literature#Klaassen1994|Klaassen 1994]]). This can be avoided by adding metal-salts (ZnCl, CuCl) to cells expressing the protein.<br />
<br />
=Metals=<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) ([[Team:Groningen/Literature#Ngu2006 |Ngu 2006]]) and fMT (the seaweed species ''Fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity ([[Team:Groningen/Literature#Singh2008 |Singh 2008]]), another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
===ArsR===<br />
ArsR is a trans-acting repressor that senses environmental As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and discriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified, its size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co-workers overexpressed ArsR in <i>E. coli</i> JM109 cells and found that the specific AS(III) content was 13-fold higher than the control without ArsR expression. High level expression of ArsR appeared to be toxic as a 3-fold reduction in cell density was observed. It has been shown that fusion partners reduce the toxicity of overexpression. Originally, Chen and co-workers made a fusion between ArsR and ELP (elastin protein), which is build out of VPGVG repeats. Because making a ArsR ELP153 fusion is very time consuming, we choose to make a fusion between MBP (maltose binding protein) and ArsR ([[Team:Groningen/Literature#Chen1998|Chen 1998]]).<br />
<BR><br />
Also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT was not successful, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
====Results====<br />
<br />
The construct MBP ArsR was built by<br />
<br />
===fMT===<br />
The <i>Fucus</i> [http://www.bioc.uzh.ch/mtpage/intro.html Metallothionein] (fMT) was isolated from the [http://en.wikipedia.org/wiki/Seaweed macroalgae] [http://en.wikipedia.org/wiki/Fucus_vesiculosus <i>Fucus vesiculosus</i> ]([[Team:Groningen/Literature#Morris1999|Morris 1999]]). It consists of 67 amino acid residues and has 16 cysteine residues, a high cysteine content is a key feature of MT. Another characteristic is the lack of aromatic residues is also seen in fMT where it only has one, tryptophan. Two domains containing cysteine residues are presumed to be involved in the metal binding function. Unusual in fMT is the presence of a 14 amino acid linker region between the two putative metal-binding domains which contains no cysteine residues. Plant MTs show this feature with about 40 residues, where vertebrate MTs only have three residues ([[Team:Groningen/Literature#Morris1999|Morris 1999]]). Being a MT fMT binds a multitude of metal ions, 6 Cd<sup>2+</sup> ions or 5 As<sup>3+</sup> ions in a sequential order, facilitated by the elongated linker domain {{todo|BUSY}}<br />
<br />
<br />
====Results====<br />
Arsenite uptake [[Team:Groningen/Protocols|assays]] were done to determine the As(III) accumulation of ''E. coli'' WT and fMT / GlpF overexpression strains. The concentration was measured by [[Team:Groningen/Protocols|ICP-MS]]. <br />
<br />
The arsenic uptake in ''E. coli'' WT (figure 3) as measured during this project (by [http://www.rikilt.wur.nl/NL/ RIKILT], Wageningen University), was compared with the uptake of ''E. coli'' with ArsR overexpression (described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], see figure 3). This shows that the arsenic uptake in ''E. coli'' WT behaves similar but has lower final As(III) uptake yield. The difference is about 10% in the standard mode, but a higher extracellular arsenic concentration seems to be needed to saturate the uptake of arsenic in ''E. coli'' WT compared to ''E. coli'' with ArsR overexpression. This can be seen by comparing the transition point to saturation in figure 3, which are respectively around 50µM As(III) and around 20µM. <br />
<br />
[[Image:As uptake in E coli ArsR overexp - Kostal 2004.PNG]]<br />
:Figure 3: Uptake of As(III) by ''E. coli'' WT (containing pSB1A2-pLac)<br />
<br />
There is a relatively large difference between the data generated by measuring the arsenic concentration with ICP-MS in the standard mode and measuring in the collusion cell technology mode (CCT mode). The difference between these two techniques is that in the standard mode it is possible that multi-atomic compounds lead to interference with the arsenic (mw = 75) peak, like argon-chloride (Ar = 40 + Cl = 35 (75%) or 37(25%)). Because 25% of this compound is found in the mw = 77 peak, a correction factor may be calculated to correct for this, the ICP-MS software (Thermo) automatically corrects for Ar-Cl interference. It uses the amount of Krypton and Selenium for this correction. In the CCT all multi-atomic compounds are supposed to be decomposed, therefore no interference will be found in this mode. But a disadvantage of this mode is that the resolution is 10x lower than the standard mode, leading to a smaller signal-to-noise ratio. Because of this, we decided to use the standard mode (corrected for interference) to determine the arsenic accumulation by ''E. coli''.<br />
<br />
A second arsenic measurement was performed (by [http://www.vwa.nl/portal/page?_pageid=119,1639634&_dad=portal&_schema=PORTAL Food and Consumer Product Safety Authority], Groningen) using ''E. coli'' WT and ''E. coli'' containing the [accumulation device] (<partinfo>BBa_K190038</partinfo>) and the different parts ([[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>)). The data was measured in the standard mode and the calculated arsenic imported by the cells is shown in figure 4.<br />
<br />
[[Image:As_uptake_in_WT_fMT_GlpF_ArsR.PNG]]<br />
:Figure 4: Uptake of As(III) by ''E. coli'' WT, and the strains containing the different parts of the accumulation device. As a control the arsenic uptake of ''E. coli'' with ArsR overexpression (as described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]]) is also shown.<br />
<br />
The curves in this figure show that there is no difference between the arsenic uptake by ''E. coli'' WT and by ''E. coli'' plus (parts of) the accumulation device. As a second observation, it can be seen that the uptake of arsenic in measured here is higher than found before (figure 3). A ratio of 2-3x was found for the WT strains (pSB1A2 and pArsR-RFP). These two differences will be discussed below. fMT shows exceptionally low arsenite uptake, this may be caused by incidentally "burning" the already dried cells at ~100;deg&C.<br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
====Discussion====<br />
Between the two data sets there are a few differences, first there seems to be no difference between arsenic uptake in WT and ''E. coli'' with the accumulation device (or parts of this). Secondly, the data of arsenic uptake by ''E. coli'' WT could was not reproducible and the last data set showed a arsenic uptake which was even higher for ''E. coli'' WT than the ''E. coli'' ArsR overexpression strain. <br />
<br />
*Why is there no difference between the ''E. coli'' WT and the ''E. coli'' with accumulation device?<br />
This can be caused by non-functional expression of one of the genes (fMT or GlpF) or both. For membrane proteins it is known that functional overexpression is harder than for cytoplasmic proteins ([[Team:Groningen/Literature#Lundstom2006|Lundstom 2006]]). This could be tested by doing As(III) uptake/binding experiments with purified proteins, but this requires protein purification which could be facilitated by the addition of a his-tag (not present yet). The function of the transporter can be tested by measuring the uptake in membrane vesicles and that of the accumulation protein can be tested by measuring metal binding for instance by isothermal titration calorimetry. Otherwise the proteins may not be produced at all, this should be tested by protein purification or sds-page. Another possibility is that these proteins cannot be produced by ‘’E. coli’’ at once, though functional expression was already proven by Singh ''et al.'' ([[Team:Groningen/Literature#Singh2008|Singh 2008]]).<br />
<br />
*Non reproducible concentrations of arsenic, imported by ''E. coli'' WT, which can be seen as there is a large difference (2-3x) in arsenic uptake determined from the first and the second measurement. All data from the second ICP-MS arsenic determination, were also unexpectedly higher than was found in literature ([[Team:Groningen/Literature#Kostal2004|Kostal 2004]], [[Team:Groningen/Literature#Singh2008|Singh 2008]]). This discrepancy may be caused by one of the following reasons.<br />
During the second arsenic uptake assay the time between the incubation and washing the cells was decreased to the minimum though during the first assay there was some time for the cells to export the As(III) via there exporter ArsB. This may have caused the lower uptake yield of arsenic in the first data set. Also there was a difference in cell concentration, in the second assay this was 2.5 times higher. It is presumably that with a higher cell concentration the uptake rate is slower but a saturating incubation time (>1hr) might cause that the equilibrium of arsenite concentration in/outside the cell is reached faster. After destruction of the samples of the first data set, the samples did not become a clear solution but a suspension containing white flakes. These were removed by centrifugation, but this seems to indicate incomplete destruction. This was not seen for the second samples, therefore an increased arsenite concentration may be measured as arsenite bound to the white flakes is not measured. <br />
It also might be, that during the second arsenite uptake assay the cells were washed less properly causing the concentration to become way higher than the first measurement. A more acidic buffer used for washing the cells is probably more efficient in removing metal ions than the TB74S buffer (pH 7.4), but as this protocol was the same as described by [[Team:Groningen/Literature#Kostal2004|Kostal 2004]], this should be a major problem. The expected increase in arsenic concentration should be linear with the external arsenite concentration, but this was not seen (figure 4), a clear saturation curve was seen. <br />
A plausible cause is that there was a mistake in the calculations, a correction factor which was forgotten to correct for. Another plausible cause is that the concentration is higher because the measured concentration was for some samples 5 times higher than the calibration range. It might be that linear extrapolation is not correct. This can cause the structural increased arsenic uptake. <br />
<br />
*Other considerations:<br />
-Metal buffer interactions, causing a lower free-As(III) concentration surrounding the cell suspension.<br />
-Arsenic oxidation in aerobic conditions to As(V), this equilibrium may change over hours, so if the stock solution is enriched with As(V) it may take hours before it is changed to As(III) again. <br />
- Binding of other metal ions to the metallothionein causing competition for arsenite binding to fMT. Possible metal ions can be: Copper(I) or other metal ions present in the undefined LB medium. A requiry is that the metal ion should bind stronger or as strong to the MT as arsenite, which binds less strongly to MT than Zn(II) for instance or Cu(I).<br />
<br />
==Copper==<br />
<br />
===MymT===<br />
<br />
MymT is a 5kDa-protein which binds Cu(I) but also to less extend Zn(II) from ''Mycobaterterium tuberculosis''. This MT was found to bind 4-6 Cu(I) ions per molecule. Induction of the expression of MymT is the strongest with Cd and Cu. But upon over-expression of MymT in ''E. coli'', the protein becomes insoluble ([[Team:Groningen/Literature#Gold2008|Gold 2008]]). This may be caused by the fact that it is a protein from a gram-positive bacteria expressed in a gram negative bacterium. Therefore specialized cultivation conditions are needed, the cells should be grown at a low temperature (16 &deg;C). The functionality of MymT can be measured by fluorescence spectroscopy, as also found for other copper binding metallothioneins. Copper bound to MT create Cu-thiolates which can be excited at 280nm and gives a Stokes shift towards 600 nm ([[Team:Groningen/Literature#Beltramini1981|Beltramini 1981]]). <br />
<br />
'''Results:'''<br />
PCR on ''mym''T from pGB68 unfortunately did not give any correct cDNA fragments, even though the primer quality was improved ([[Team:Groningen/Protocols|Protocol Biobrick primers]]). Therefore the sub-project was discontinued.<br />
<br />
==Zinc==<br />
Below toxic concentrations, zinc is essential for many biological processes. Examples are enzymatic hydroxylation, DNA and RNA synthesis, transcription and translation, signal transduction and apoptosis regulation ([[http://en.wikipedia.org/wiki/Zinc 1]] and [[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]). Methallothioneins can adjust the zinc absorption up to 14-40%, though a real excess of zinc can be toxic. A daily intake of 100–300 mg Zn/day can give rise to copper / iron deficiency and damage of nerve receptors ([[Team:Groningen/Literature#Fosmire1990|Fosmire 1990]]). Examples of metallothioneins sequestering zinc, are SmtA from the cyanobacterium ''Synechococcus'' PCC7942 ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]), ZiaR from ''Synechocystis'' PCC 6803 ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]), human metallothioneins like MT-1 and -2. The mammalian proteins were found to bind 7 Zn<sup>2+</sup> ions by the thiolate-group of there cysteins.<br />
<br />
===SmtA===<br />
SmtA is a MT from ''Synechococcus'' PCC 6803, it was found to bind 3-4Zn ions and is supposed to have a function in preventing zinc toxicity ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]), but it also binds copper and cadmium ([[Team:Groningen/Literature#Shi1992|Shi 1992]]). Upon binding of Zn, the glutathione transferase fusion-protein showed a 1:3 stoichiometry and SmtA a 1:4 stoichiometry ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]). SmtA binds the 4 Zn ions via cystein thiolate-bridges, forming a Zn<sub>4</sub>Cys<sub>11</sub> cluster whichs was also found in mammalian MT, though these proteins do not have a homologous DNA sequence ([[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]]). ''SmtA'' is found on an operon with it's transcriptional regulator ''smtB''. SmtB releases from the promoter-operator region in front of this operon, when it binds Zn via its metal binding motif. SmtB and [[Team:Groningen/Project/Accumulation#Arsenic|ArsR]] (negative transcriptional regulator binding arsenic) have similar functionalities but differ in metal binding motifs ([[Team:Groningen/Literature#Robinson2001|Robinson 2001]]). That ''Synechococcus'' is a gram negative bacterium might increase the possibility of functional and stable overexpression in ''E. coli'' .<br />
<br />
===Results===<br />
PCR reactions to amplify SmtA from pET29a and SmtA-GST from pGEX-3x were successful, but unfortunately the DNA sequence used to design the SmtA primers was not correct so therefore wrong cDNA fragments were amplified. Because it was too late to order new primers, this sub-project was discontinued.<br />
<br />
==Alternatives==<br />
{{todo|Inclusion bodies}} ([[Team:Groningen/Literature#Fowler1987|Fowler 1987]])<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
<!--==Inhibitory characteristics?==--><br />
<br />
==Modelling==<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
{{GraphHeader}}<br />
===Arsenic - ArsR===<br />
<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add up to a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal 2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Read inputs<br />
var awAs = getInput('awAs'); // g/mol<br />
var cAs = getInput('cAs') * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = getInput('Mcelldrywet'); // kg/kg<br />
var rhocell = getInput('rhocell'); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set outputs<br />
setOutput('Aspercellvolume', Aspercellvolume);<br />
setOutput('molAspercellvolume', molAspercellvolume);<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|frame]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right, a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
In addition to binding to As(III), ArsR can repress Ars, creating a negative feedback loop. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen 1997]]). In the <i>E. coli</i> top 10 there is only ars promoter present on the genome to produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD, but these are not used in this project. We intend to introduce instead a constitutive promoter (pro), which produces just ArsR, in order to bind as much As(III) as possible.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary.<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
KR<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
<nobr>KA<sub>d</sub> (ArsR<sub>ars</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)</nobr><br/><br />
KM<sub>d</sub> (MBPArsR<sub>As</sub>) = <input type="text" id="KMd" value="6"/> &micro;M (???)<br/><br />
KF<sub>d</sub> (fMTArsR<sub>As</sub>) = <input type="text" id="KFd" value="6"/> &micro;M (???)<br/><br />
n<sub>f</sub> = <input type="text" id="nf" value="3"/> (???)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;R (ArsR) = <input type="text" id="tauR" value="0.1"/> min (???)<br/><br />
&tau;M (MBPArsR) = <input type="text" id="tauM" value="0.1"/> min (???)<br/><br />
&tau;F (fMT) = <input type="text" id="tauF" value="0.1"/> min (???)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
<nobr>&beta;RN (ars1 &rarr; ArsR) = <input type="text" id="beta1" value="100"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;R (proR &rarr; ArsR) = <input type="text" id="beta3" value="100"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;M (proM &rarr; MBPArsR) = <input type="text" id="betaM" value="26.6"/> 1/second (???)</nobr><br/><br />
<nobr>&beta;F (proF &rarr; fMT) = <input type="text" id="betaF" value="200"/> 1/second (???)</nobr><br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
ars1<sub>total</sub> = <input type="text" id="ars1TPerCell" value="1"/> per cell<br/><br />
<nobr>proR = <input type="text" id="proRPerCell" value="0"/> per cell (??)</nobr><br/><br />
<nobr>proM = <input type="text" id="proMPerCell" value="100"/> per cell (??)</nobr><br/><br />
<nobr>proF = <input type="text" id="proFPerCell" value="0"/> per cell (??)</nobr><br/><br />
V<sub>cell</sub> = <input type="text" id="Vcell" value="1"/> &micro;m<sup>3</sup> </html><br />
([http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| CCBD])<html><br />
<br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>ArsR</dt><br />
<dd><br />
ars / ars<sub>total</sub> = <span id="arsFraction"></span><br/><br />
ArsR = <span id="ArsR"></span> &micro;M<br/><br />
<!--ArsR<sub>total</sub> = <span id="ArsRT"></span> &micro;M<br/>--><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="AsinT"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="AsinTfactor"></span><br/><br />
</dd><br />
</dl><br />
</html><br />
<span id="accumulationFactorData"></span><br />
{{graph|Team:Groningen/Graphs/AccumulationFactor|id=accumulationFactorGraph}}<br />
(For constants other than the ones on the left the [[Team:Groningen/Modelling/Arsenic.js|default values]] are used.)<br />
<html><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Intermediates (mostly useful for debugging)<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Read inputs<br />
var c = arsenicModelConstants();<br />
c.AsT = 0;<br />
c.K1d = getInput('K1d') * 1e-6; // micromolar -> molar<br />
c.K3d2 = Math.pow(getInput('K3d') * 1e-6,2); // micromolar -> molar<br />
c.KMd = getInput('KMd') * 1e-6; // micromolar -> molar<br />
c.KFd = getInput('KFd') * 1e-6; // micromolar -> molar<br />
c.nf = getInput('nf') * 1e-6; // micromolar -> molar<br />
c.tauR = getInput('tauR') * 60; // minutes -> seconds<br />
c.tauM = getInput('tauM') * 60; // minutes -> seconds<br />
c.tauF = getInput('tauF') * 60; // minutes -> seconds<br />
c.beta1 = getInput('beta1'); // 1/second<br />
c.beta3 = getInput('beta3'); // 1/second<br />
c.betaM = getInput('betaM'); // 1/second<br />
c.betaF = getInput('betaF'); // 1/second<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var Vcell = getInput('Vcell') * 1e-15; // micrometer^3/cell -> liter/cell<br />
c.ars1T = getInput('ars1TPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.ars2T = 0;<br />
c.pro = getInput('proRPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.proM = getInput('proMPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
c.proF = getInput('proFPerCell') / (avogadro*Vcell); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var x = arsenicModelEquilibrium(c);<br />
var ArsR = x._ArsR;<br />
var arsFraction = x._arsF;<br />
var AsinTfactor = 1 + ArsR/c.K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
setOutput('arsFraction', arsFraction);<br />
setOutput('ArsR', ArsR * 1e6);<br />
<br />
// Set outputs<br />
setOutput('AsinTfactor', AsinTfactor);<br />
<br />
// Draw graph<br />
var dataNode = document.getElementById("accumulationFactorData");<br />
var graphNode = document.getElementById("accumulationFactorGraph");<br />
var data = {AsT:[], AsexT0:[], AsinT:[], AsinT2:[]};<br />
var bAsin, cAsin, Asin;<br />
// Some guesses<br />
var c2 = {}, x2;<br />
for(var a in c) c2[a] = c[a];<br />
c2.v5 *= 2;<br />
for(var AsexT0uM=0.1; AsexT0uM<=100; AsexT0uM*=1.1) {<br />
c.AsT = c.Vs*AsexT0uM*1e-6;<br />
c2.AsT = c.AsT;<br />
x = arsenicModelEquilibrium(c);<br />
x2 = arsenicModelEquilibrium(c2);<br />
data.AsT.push(c.AsT);<br />
data.AsexT0.push(c.AsT/c.Vs);<br />
data.AsinT.push(x.AsinT*c.Vc/c.AsT);<br />
data.AsinT2.push(x2.AsinT*c.Vc/c.AsT);<br />
}<br />
dataNode.data = data;<br />
if (graphNode.refresh) graphNode.refresh();<br />
}<br />
</script><br />
</html><br />
<br />
'''In conclusion:'''<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it should not matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for Ars promotors, you can see that it is clearly advantageous to use constitutive promotors as just adding ars promoters does not increase the accumulation factor. A plasmid containing an ars promoter and (just) a gene coding for ArsR behind it might contain more arsenic, but there would also be more unbound arsenic, increasing the toxicity.<br />
* The model is not very sensitive to different values for K3d (with K3d=1mM the accumulation factor is 248.06 and with K3d=1nM it is 240.57).<br />
* The accumulation factor is greatly affected by the product of the half-life of ArsR and the production rate.<br />
<br />
<!-- ==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies [[Team:Groningen/Literature#Lin2007-2|Lin 2007]]<br />
**** Inductively coupled mass spectrometry (ICP-MS) ([[Team:Groningen/Literature#Meng2004|Meng 2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen 1997]]). In the paper ArsD is purified, but if that is not feasible for us, we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest--><br />
{{Team:Groningen/Project/Footer}}</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-09-11T10:50:19Z<p>Frans: /* Arsenic */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
==Introduction==<br />
<br />
Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..<br />
<br />
===Metallothioneins===<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) '''({{todo|Merrifield2004}})'''. And they have readily been used to create cell based systems for purification of contaminated water [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Brady1994|Brady1994]]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one [[Team:Groningen/Literature#Chang1998|Chang1998]].<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium [[Team:Groningen/Literature#Deng2007|Deng2007]], arsenic [[Team:Groningen/Literature#Ngu2006|Ngu2006]], [[Team:Groningen/Literature#Kostal2004|Kostal2004]], [[Team:Groningen/Literature#Singh2008|Singh2008]], mercury [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Chen1998|Chen1997-2]], [[Team:Groningen/Literature#Deng2008|Deng2008]], nickel [[Team:Groningen/Literature#Deng2003|Deng2003]] or a combination of metals [[Team:Groningen/Literature#Chang1998|Chang1998]], [[Team:Groningen/Literature#Kao2008|Kao2008]].<br />
Metal-protein complexes can be quantified using a fluorescent molecule [[Team:Groningen/Literature#Cadosch2008 |Cadosch2008]].<br />
<br />
===Cloning strategy===<br />
{{todo}}[[Image:Cloning MT Transporter in AC3-new.pdf]]<br />
<br />
===Metals===<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) [[Team:Groningen/Literature#Ngu2006 |Ngu2006]] and fMT (the seaweed species ''fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity [[Team:Groningen/Literature#Singh2008 |Singh2008]], another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
ArsR is a trans-acting repressor that senses environmetal As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified it's size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co workers overexpressed ArsR in E.Coli JM109 cells and found that the specific AS(III) content was 13 fold higher than the control without arsR expression. High level expression of ArsR appeared to be toxic as a 3 fold reduction in cell density was observed. It has been shown that fusion partners reduce the toxicity of overexpression. Originally Chen and co workers made a fusion between ArsR and ELP(elastin protein) which is build out of VPGVG repeats. Because making a ArsR ELP153 fusion is very time consuming, we choose to make a fusion between MBP (maltose binding protein) and ArsR. [[Team:Groningen/Literature#Chen1998|Chen1998]]<br />
<br />
<br />
also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT wasn't succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
==Copper==<br />
pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) [[Team:Groningen/Literature#Gold2008 |Gold2008]]<br />
**Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).<br />
<br />
==Zinc==<br />
*Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) [[Team:Groningen/Literature#Blindauer2001|Blindauer2001]]<br />
<br />
===Alternatives===<br />
{{todo|Inclusion bodies}} [[Team:Groningen/Literature#Fowler1987 |Fowler1987]]<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
===Inhibitory characteristics?===<br />
<br />
==Modelling==<br />
===Arsenic - ArsR===<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Input<br />
var awAsNode = document.getElementById("awAs");<br />
var cAsNode = document.getElementById("cAs");<br />
var McelldrywetNode = document.getElementById("Mcelldrywet");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var AspercellvolumeNode = document.getElementById("Aspercellvolume");<br />
var molAspercellvolumeNode = document.getElementById("molAspercellvolume");<br />
<br />
// Read inputs<br />
var awAs = Number(awAsNode.value); // g/mol<br />
var cAs = Number(cAsNode.value) * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = Number(McelldrywetNode.value); // kg/kg<br />
var rhocell = Number(rhocellNode.value); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(AspercellvolumeNode, Aspercellvolume);<br />
setOutput(molAspercellvolumeNode, molAspercellvolume);<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|thumb|In addition to binding to As(III), ArsR can repress expression of OpG. This is a negative feedback to the operon. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen1997]]).<br />
In the <i>E. coli</i> top10 there is only OpG present on the genome, which produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD (not used in this project). We intend to introduce instead OpH, which constitutively produces ArsR, in order to produce an abundance of ArsR.<br />
]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. {{todo|TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?}}<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
K1<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
K3<sub>d</sub> (ArsR<sub>opn</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;1 (ArsR) = <input type="text" id="tau1" value="30"/> min (??, dilution)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
&beta;1 (ArsR) = <input type="text" id="beta1" value="1000"/> 1/second (???)<br/><br />
&beta;3 (ArsR constitutive) = <input type="text" id="beta3" value="1000"/> 1/second (???)<br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
OpG<sub>total</sub> = <input type="text" id="OpGTotalPerCell" value="1"/> per cell (?)<br/><br />
OpH = <input type="text" id="OpHPerCell" value="10"/> per cell (??)<br/><br />
V<sub>cell</sub> = <input type="text" id="Vc" value="1"/> &micro;m<sup>3</sup> </html>[http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi]<html><br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>Unbound substances</dt><br />
<dd><br />
OpG / OpG<sub>total</sub> = <span id="OpGFraction"></span><br/><br />
ArsR = <span id="ArsRConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>Bound substances</dt><br />
<dd><br />
<!--ArsR<sub>As</sub> = <span id="ArsRAs3Concentration"></span> &micro;M<br/>--><br />
ArsR<sub>op</sub> = <span id="ArsROpConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="As3TotalConcentration"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="As3TotalFactor"></span><br/><br />
</dd><br />
</dl><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Input<br />
var K1dNode = document.getElementById("K1d");<br />
var K3dNode = document.getElementById("K3d");<br />
var tau1Node = document.getElementById("tau1");<br />
var beta1Node = document.getElementById("beta1");<br />
var beta3Node = document.getElementById("beta3");<br />
//var As3Node = document.getElementById("As3Concentration");<br />
var OpGTPerCellNode = document.getElementById("OpGTotalPerCell");<br />
var OpHPerCellNode = document.getElementById("OpHPerCell");<br />
var VcNode = document.getElementById("Vc");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var OpGFractionNode = document.getElementById("OpGFraction");<br />
var ArsRNode = document.getElementById("ArsRConcentration");<br />
//var ArsRAs3Node = document.getElementById("ArsRAs3Concentration");<br />
var ArsROpNode = document.getElementById("ArsROpConcentration");<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Outputs<br />
//var As3TNode = document.getElementById("As3TotalConcentration");<br />
var As3TFactorNode = document.getElementById("As3TotalFactor");<br />
<br />
// Read inputs<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var K1d = Number(K1dNode.value) * 1e-6; // micromolar -> molar<br />
var K3d2 = Math.pow(Number(K3dNode.value) * 1e-6,2); // micromolar -> molar<br />
var tau1 = Number(tau1Node.value) * 60; // minutes -> seconds<br />
var beta1 = Number(beta1Node.value); // 1/second<br />
var beta3 = Number(beta3Node.value); // 1/second<br />
//var As3 = Number(As3Node.value) * 1e-6; // micromolar -> molar<br />
var Vc = Number(VcNode.value) * 1e-15; // micrometer^3/cell -> liter/cell<br />
var OpGT= Number(OpGTPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
var OpH = Number(OpHPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
// Fixed point iteration<br />
var ArsR= 1;<br />
do {<br />
var fx = Math.pow(ArsR,3) - (tau1/Math.LN2)*beta3*OpH*Math.pow(ArsR,2) + K3d2*ArsR<br />
- K3d2*(tau1/Math.LN2)*(beta1*OpGT + beta3*OpH);<br />
var dfx = 3*Math.pow(ArsR,2) - 2*(tau1/Math.LN2)*beta3*OpH*ArsR + K3d2;<br />
var ddfx = 6*ArsR - 2*(tau1/Math.LN2)*beta3*OpH;<br />
ArsR = ArsR - 2*fx*dfx/(2*Math.pow(dfx,2)-fx*ddfx);<br />
} while(Math.abs(fx)>1e-6);<br />
<br />
var OpG = OpGT/(Math.pow(ArsR,2)/K3d2 + 1);<br />
//var ArsRAs3 = ArsR * As3 / K1d;<br />
var ArsROp = Math.pow(ArsR,2) * OpG / K3d2;<br />
<br />
var As3TFactor = 1 + ArsR/K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (OpGFractionNode) setOutput(OpGFractionNode, OpG/OpGT);<br />
if (ArsRNode) setOutput(ArsRNode, ArsR* 1e6);<br />
//if (ArsRAs3Node) setOutput(ArsRAs3Node, ArsRAs3* 1e6);<br />
if (ArsROpNode) setOutput(ArsROpNode, ArsROp* 1e6);<br />
<br />
// Set outputs<br />
//setOutput(As3TNode, As3Total* 1e6);<br />
setOutput(As3TFactorNode, As3TFactor);<br />
}<br />
</script><br />
</html><br />
<br />
In conclusion:<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors (they give a much higher increase in accumulation).<br />
* The model is not very sensitive to different values for K3d (with K3d=1M the accumulation factor is 7905.0 and with K3d=10<sup>-12</sup>M it is 7188.0).<br />
<br />
==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies ([[Team:Groningen/Literature#Lin2007-2|Lin2007]])<br />
**** Inductively coupled mass spectrometry (ICP-MS) (([[Team:Groningen/Literature#Meng2004|Meng2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen1997]]). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-09-11T10:21:08Z<p>Frans: /* Arsenic */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
==Introduction==<br />
<br />
Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..<br />
<br />
===Metallothioneins===<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) '''({{todo|Merrifield2004}})'''. And they have readily been used to create cell based systems for purification of contaminated water [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Brady1994|Brady1994]]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one [[Team:Groningen/Literature#Chang1998|Chang1998]].<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium [[Team:Groningen/Literature#Deng2007|Deng2007]], arsenic [[Team:Groningen/Literature#Ngu2006|Ngu2006]], [[Team:Groningen/Literature#Kostal2004|Kostal2004]], [[Team:Groningen/Literature#Singh2008|Singh2008]], mercury [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Chen1998|Chen1997-2]], [[Team:Groningen/Literature#Deng2008|Deng2008]], nickel [[Team:Groningen/Literature#Deng2003|Deng2003]] or a combination of metals [[Team:Groningen/Literature#Chang1998|Chang1998]], [[Team:Groningen/Literature#Kao2008|Kao2008]].<br />
Metal-protein complexes can be quantified using a fluorescent molecule [[Team:Groningen/Literature#Cadosch2008 |Cadosch2008]].<br />
<br />
===Cloning strategy===<br />
{{todo}}[[Image:Cloning MT Transporter in AC3-new.pdf]]<br />
<br />
===Metals===<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) [[Team:Groningen/Literature#Ngu2006 |Ngu2006]] and fMT (the seaweed species ''fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity [[Team:Groningen/Literature#Singh2008 |Singh2008]], another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
ArsR is a trans-acting repressor that senses environmetal As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified it's size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co workers overexpressed ArsR in E.Coli JM109 cells and found that the over 38 h the concentration As(III) 20 fold. Th[[Team:Groningen/Literature#Chen1998|Chen1998]]<br />
<br />
<br />
also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT wasn't succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
==Copper==<br />
pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) [[Team:Groningen/Literature#Gold2008 |Gold2008]]<br />
**Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).<br />
<br />
==Zinc==<br />
*Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) [[Team:Groningen/Literature#Blindauer2001|Blindauer2001]]<br />
<br />
===Alternatives===<br />
{{todo|Inclusion bodies}} [[Team:Groningen/Literature#Fowler1987 |Fowler1987]]<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
===Inhibitory characteristics?===<br />
<br />
==Modelling==<br />
===Arsenic - ArsR===<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Input<br />
var awAsNode = document.getElementById("awAs");<br />
var cAsNode = document.getElementById("cAs");<br />
var McelldrywetNode = document.getElementById("Mcelldrywet");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var AspercellvolumeNode = document.getElementById("Aspercellvolume");<br />
var molAspercellvolumeNode = document.getElementById("molAspercellvolume");<br />
<br />
// Read inputs<br />
var awAs = Number(awAsNode.value); // g/mol<br />
var cAs = Number(cAsNode.value) * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = Number(McelldrywetNode.value); // kg/kg<br />
var rhocell = Number(rhocellNode.value); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(AspercellvolumeNode, Aspercellvolume);<br />
setOutput(molAspercellvolumeNode, molAspercellvolume);<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|thumb|In addition to binding to As(III), ArsR can repress expression of OpG. This is a negative feedback to the operon. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen1997]]).<br />
In the <i>E. coli</i> top10 there is only OpG present on the genome, which produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD (not used in this project). We intend to introduce instead OpH, which constitutively produces ArsR, in order to produce an abundance of ArsR.<br />
]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. {{todo|TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?}}<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
K1<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
K3<sub>d</sub> (ArsR<sub>opn</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;1 (ArsR) = <input type="text" id="tau1" value="30"/> min (??, dilution)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
&beta;1 (ArsR) = <input type="text" id="beta1" value="1000"/> 1/second (???)<br/><br />
&beta;3 (ArsR constitutive) = <input type="text" id="beta3" value="1000"/> 1/second (???)<br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
OpG<sub>total</sub> = <input type="text" id="OpGTotalPerCell" value="1"/> per cell (?)<br/><br />
OpH = <input type="text" id="OpHPerCell" value="10"/> per cell (??)<br/><br />
V<sub>cell</sub> = <input type="text" id="Vc" value="1"/> &micro;m<sup>3</sup> </html>[http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi]<html><br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>Unbound substances</dt><br />
<dd><br />
OpG / OpG<sub>total</sub> = <span id="OpGFraction"></span><br/><br />
ArsR = <span id="ArsRConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>Bound substances</dt><br />
<dd><br />
<!--ArsR<sub>As</sub> = <span id="ArsRAs3Concentration"></span> &micro;M<br/>--><br />
ArsR<sub>op</sub> = <span id="ArsROpConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="As3TotalConcentration"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="As3TotalFactor"></span><br/><br />
</dd><br />
</dl><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Input<br />
var K1dNode = document.getElementById("K1d");<br />
var K3dNode = document.getElementById("K3d");<br />
var tau1Node = document.getElementById("tau1");<br />
var beta1Node = document.getElementById("beta1");<br />
var beta3Node = document.getElementById("beta3");<br />
//var As3Node = document.getElementById("As3Concentration");<br />
var OpGTPerCellNode = document.getElementById("OpGTotalPerCell");<br />
var OpHPerCellNode = document.getElementById("OpHPerCell");<br />
var VcNode = document.getElementById("Vc");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var OpGFractionNode = document.getElementById("OpGFraction");<br />
var ArsRNode = document.getElementById("ArsRConcentration");<br />
//var ArsRAs3Node = document.getElementById("ArsRAs3Concentration");<br />
var ArsROpNode = document.getElementById("ArsROpConcentration");<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Outputs<br />
//var As3TNode = document.getElementById("As3TotalConcentration");<br />
var As3TFactorNode = document.getElementById("As3TotalFactor");<br />
<br />
// Read inputs<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var K1d = Number(K1dNode.value) * 1e-6; // micromolar -> molar<br />
var K3d2 = Math.pow(Number(K3dNode.value) * 1e-6,2); // micromolar -> molar<br />
var tau1 = Number(tau1Node.value) * 60; // minutes -> seconds<br />
var beta1 = Number(beta1Node.value); // 1/second<br />
var beta3 = Number(beta3Node.value); // 1/second<br />
//var As3 = Number(As3Node.value) * 1e-6; // micromolar -> molar<br />
var Vc = Number(VcNode.value) * 1e-15; // micrometer^3/cell -> liter/cell<br />
var OpGT= Number(OpGTPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
var OpH = Number(OpHPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
// Fixed point iteration<br />
var ArsR= 1;<br />
do {<br />
var fx = Math.pow(ArsR,3) - (tau1/Math.LN2)*beta3*OpH*Math.pow(ArsR,2) + K3d2*ArsR<br />
- K3d2*(tau1/Math.LN2)*(beta1*OpGT + beta3*OpH);<br />
var dfx = 3*Math.pow(ArsR,2) - 2*(tau1/Math.LN2)*beta3*OpH*ArsR + K3d2;<br />
var ddfx = 6*ArsR - 2*(tau1/Math.LN2)*beta3*OpH;<br />
ArsR = ArsR - 2*fx*dfx/(2*Math.pow(dfx,2)-fx*ddfx);<br />
} while(Math.abs(fx)>1e-6);<br />
<br />
var OpG = OpGT/(Math.pow(ArsR,2)/K3d2 + 1);<br />
//var ArsRAs3 = ArsR * As3 / K1d;<br />
var ArsROp = Math.pow(ArsR,2) * OpG / K3d2;<br />
<br />
var As3TFactor = 1 + ArsR/K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (OpGFractionNode) setOutput(OpGFractionNode, OpG/OpGT);<br />
if (ArsRNode) setOutput(ArsRNode, ArsR* 1e6);<br />
//if (ArsRAs3Node) setOutput(ArsRAs3Node, ArsRAs3* 1e6);<br />
if (ArsROpNode) setOutput(ArsROpNode, ArsROp* 1e6);<br />
<br />
// Set outputs<br />
//setOutput(As3TNode, As3Total* 1e6);<br />
setOutput(As3TFactorNode, As3TFactor);<br />
}<br />
</script><br />
</html><br />
<br />
In conclusion:<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors (they give a much higher increase in accumulation).<br />
* The model is not very sensitive to different values for K3d (with K3d=1M the accumulation factor is 7905.0 and with K3d=10<sup>-12</sup>M it is 7188.0).<br />
<br />
==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies ([[Team:Groningen/Literature#Lin2007-2|Lin2007]])<br />
**** Inductively coupled mass spectrometry (ICP-MS) (([[Team:Groningen/Literature#Meng2004|Meng2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen1997]]). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-09-11T10:00:51Z<p>Frans: /* Arsenic */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
==Introduction==<br />
<br />
Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..<br />
<br />
===Metallothioneins===<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) '''({{todo|Merrifield2004}})'''. And they have readily been used to create cell based systems for purification of contaminated water [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Brady1994|Brady1994]]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one [[Team:Groningen/Literature#Chang1998|Chang1998]].<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium [[Team:Groningen/Literature#Deng2007|Deng2007]], arsenic [[Team:Groningen/Literature#Ngu2006|Ngu2006]], [[Team:Groningen/Literature#Kostal2004|Kostal2004]], [[Team:Groningen/Literature#Singh2008|Singh2008]], mercury [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Chen1998|Chen1997-2]], [[Team:Groningen/Literature#Deng2008|Deng2008]], nickel [[Team:Groningen/Literature#Deng2003|Deng2003]] or a combination of metals [[Team:Groningen/Literature#Chang1998|Chang1998]], [[Team:Groningen/Literature#Kao2008|Kao2008]].<br />
Metal-protein complexes can be quantified using a fluorescent molecule [[Team:Groningen/Literature#Cadosch2008 |Cadosch2008]].<br />
<br />
===Cloning strategy===<br />
{{todo}}[[Image:Cloning MT Transporter in AC3-new.pdf]]<br />
<br />
===Metals===<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) [[Team:Groningen/Literature#Ngu2006 |Ngu2006]] and fMT (the seaweed species ''fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity [[Team:Groningen/Literature#Singh2008 |Singh2008]], another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
ArsR is a trans-acting repressor that senses environmetal As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified it's size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co workers overexpressed ArsR in E.Coli JM109 cells [[Team:Groningen/Literature#Chen1998|Chen1998]]<br />
<br />
<br />
also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT wasn't succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
==Copper==<br />
pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) [[Team:Groningen/Literature#Gold2008 |Gold2008]]<br />
**Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).<br />
<br />
==Zinc==<br />
*Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) [[Team:Groningen/Literature#Blindauer2001|Blindauer2001]]<br />
<br />
===Alternatives===<br />
{{todo|Inclusion bodies}} [[Team:Groningen/Literature#Fowler1987 |Fowler1987]]<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
===Inhibitory characteristics?===<br />
<br />
==Modelling==<br />
===Arsenic - ArsR===<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Input<br />
var awAsNode = document.getElementById("awAs");<br />
var cAsNode = document.getElementById("cAs");<br />
var McelldrywetNode = document.getElementById("Mcelldrywet");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var AspercellvolumeNode = document.getElementById("Aspercellvolume");<br />
var molAspercellvolumeNode = document.getElementById("molAspercellvolume");<br />
<br />
// Read inputs<br />
var awAs = Number(awAsNode.value); // g/mol<br />
var cAs = Number(cAsNode.value) * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = Number(McelldrywetNode.value); // kg/kg<br />
var rhocell = Number(rhocellNode.value); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(AspercellvolumeNode, Aspercellvolume);<br />
setOutput(molAspercellvolumeNode, molAspercellvolume);<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|thumb|In addition to binding to As(III), ArsR can repress expression of OpG. This is a negative feedback to the operon. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen1997]]).<br />
In the <i>E. coli</i> top10 there is only OpG present on the genome, which produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD (not used in this project). We intend to introduce instead OpH, which constitutively produces ArsR, in order to produce an abundance of ArsR.<br />
]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. {{todo|TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?}}<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
K1<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
K3<sub>d</sub> (ArsR<sub>opn</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;1 (ArsR) = <input type="text" id="tau1" value="30"/> min (??, dilution)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
&beta;1 (ArsR) = <input type="text" id="beta1" value="1000"/> 1/second (???)<br/><br />
&beta;3 (ArsR constitutive) = <input type="text" id="beta3" value="1000"/> 1/second (???)<br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
OpG<sub>total</sub> = <input type="text" id="OpGTotalPerCell" value="1"/> per cell (?)<br/><br />
OpH = <input type="text" id="OpHPerCell" value="10"/> per cell (??)<br/><br />
V<sub>cell</sub> = <input type="text" id="Vc" value="1"/> &micro;m<sup>3</sup> </html>[http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi]<html><br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>Unbound substances</dt><br />
<dd><br />
OpG / OpG<sub>total</sub> = <span id="OpGFraction"></span><br/><br />
ArsR = <span id="ArsRConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>Bound substances</dt><br />
<dd><br />
<!--ArsR<sub>As</sub> = <span id="ArsRAs3Concentration"></span> &micro;M<br/>--><br />
ArsR<sub>op</sub> = <span id="ArsROpConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="As3TotalConcentration"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="As3TotalFactor"></span><br/><br />
</dd><br />
</dl><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Input<br />
var K1dNode = document.getElementById("K1d");<br />
var K3dNode = document.getElementById("K3d");<br />
var tau1Node = document.getElementById("tau1");<br />
var beta1Node = document.getElementById("beta1");<br />
var beta3Node = document.getElementById("beta3");<br />
//var As3Node = document.getElementById("As3Concentration");<br />
var OpGTPerCellNode = document.getElementById("OpGTotalPerCell");<br />
var OpHPerCellNode = document.getElementById("OpHPerCell");<br />
var VcNode = document.getElementById("Vc");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var OpGFractionNode = document.getElementById("OpGFraction");<br />
var ArsRNode = document.getElementById("ArsRConcentration");<br />
//var ArsRAs3Node = document.getElementById("ArsRAs3Concentration");<br />
var ArsROpNode = document.getElementById("ArsROpConcentration");<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Outputs<br />
//var As3TNode = document.getElementById("As3TotalConcentration");<br />
var As3TFactorNode = document.getElementById("As3TotalFactor");<br />
<br />
// Read inputs<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var K1d = Number(K1dNode.value) * 1e-6; // micromolar -> molar<br />
var K3d2 = Math.pow(Number(K3dNode.value) * 1e-6,2); // micromolar -> molar<br />
var tau1 = Number(tau1Node.value) * 60; // minutes -> seconds<br />
var beta1 = Number(beta1Node.value); // 1/second<br />
var beta3 = Number(beta3Node.value); // 1/second<br />
//var As3 = Number(As3Node.value) * 1e-6; // micromolar -> molar<br />
var Vc = Number(VcNode.value) * 1e-15; // micrometer^3/cell -> liter/cell<br />
var OpGT= Number(OpGTPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
var OpH = Number(OpHPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
// Fixed point iteration<br />
var ArsR= 1;<br />
do {<br />
var fx = Math.pow(ArsR,3) - (tau1/Math.LN2)*beta3*OpH*Math.pow(ArsR,2) + K3d2*ArsR<br />
- K3d2*(tau1/Math.LN2)*(beta1*OpGT + beta3*OpH);<br />
var dfx = 3*Math.pow(ArsR,2) - 2*(tau1/Math.LN2)*beta3*OpH*ArsR + K3d2;<br />
var ddfx = 6*ArsR - 2*(tau1/Math.LN2)*beta3*OpH;<br />
ArsR = ArsR - 2*fx*dfx/(2*Math.pow(dfx,2)-fx*ddfx);<br />
} while(Math.abs(fx)>1e-6);<br />
<br />
var OpG = OpGT/(Math.pow(ArsR,2)/K3d2 + 1);<br />
//var ArsRAs3 = ArsR * As3 / K1d;<br />
var ArsROp = Math.pow(ArsR,2) * OpG / K3d2;<br />
<br />
var As3TFactor = 1 + ArsR/K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (OpGFractionNode) setOutput(OpGFractionNode, OpG/OpGT);<br />
if (ArsRNode) setOutput(ArsRNode, ArsR* 1e6);<br />
//if (ArsRAs3Node) setOutput(ArsRAs3Node, ArsRAs3* 1e6);<br />
if (ArsROpNode) setOutput(ArsROpNode, ArsROp* 1e6);<br />
<br />
// Set outputs<br />
//setOutput(As3TNode, As3Total* 1e6);<br />
setOutput(As3TFactorNode, As3TFactor);<br />
}<br />
</script><br />
</html><br />
<br />
In conclusion:<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors (they give a much higher increase in accumulation).<br />
* The model is not very sensitive to different values for K3d (with K3d=1M the accumulation factor is 7905.0 and with K3d=10<sup>-12</sup>M it is 7188.0).<br />
<br />
==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies ([[Team:Groningen/Literature#Lin2007-2|Lin2007]])<br />
**** Inductively coupled mass spectrometry (ICP-MS) (([[Team:Groningen/Literature#Meng2004|Meng2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen1997]]). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-09-11T09:43:43Z<p>Frans: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO METAL SENSITIVE PROMOTORS" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Promoters}}</div><br />
<br />
==Introduction==<br />
<br />
Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..<br />
<br />
===Metallothioneins===<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) '''({{todo|Merrifield2004}})'''. And they have readily been used to create cell based systems for purification of contaminated water [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Brady1994|Brady1994]]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one [[Team:Groningen/Literature#Chang1998|Chang1998]].<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium [[Team:Groningen/Literature#Deng2007|Deng2007]], arsenic [[Team:Groningen/Literature#Ngu2006|Ngu2006]], [[Team:Groningen/Literature#Kostal2004|Kostal2004]], [[Team:Groningen/Literature#Singh2008|Singh2008]], mercury [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Chen1998|Chen1997-2]], [[Team:Groningen/Literature#Deng2008|Deng2008]], nickel [[Team:Groningen/Literature#Deng2003|Deng2003]] or a combination of metals [[Team:Groningen/Literature#Chang1998|Chang1998]], [[Team:Groningen/Literature#Kao2008|Kao2008]].<br />
Metal-protein complexes can be quantified using a fluorescent molecule [[Team:Groningen/Literature#Cadosch2008 |Cadosch2008]].<br />
<br />
===Cloning strategy===<br />
{{todo}}[[Image:Cloning MT Transporter in AC3-new.pdf]]<br />
<br />
===Metals===<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) [[Team:Groningen/Literature#Ngu2006 |Ngu2006]] and fMT (the seaweed species ''fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity [[Team:Groningen/Literature#Singh2008 |Singh2008]], another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
ArsR is a trans-acting repressor that senses environmetal As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified it's size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) it's a <br />
<br />
<br />
also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT wasn't succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
==Copper==<br />
pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) [[Team:Groningen/Literature#Gold2008 |Gold2008]]<br />
**Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).<br />
<br />
==Zinc==<br />
*Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) [[Team:Groningen/Literature#Blindauer2001|Blindauer2001]]<br />
<br />
===Alternatives===<br />
{{todo|Inclusion bodies}} [[Team:Groningen/Literature#Fowler1987 |Fowler1987]]<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
===Inhibitory characteristics?===<br />
<br />
==Modelling==<br />
===Arsenic - ArsR===<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Input<br />
var awAsNode = document.getElementById("awAs");<br />
var cAsNode = document.getElementById("cAs");<br />
var McelldrywetNode = document.getElementById("Mcelldrywet");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var AspercellvolumeNode = document.getElementById("Aspercellvolume");<br />
var molAspercellvolumeNode = document.getElementById("molAspercellvolume");<br />
<br />
// Read inputs<br />
var awAs = Number(awAsNode.value); // g/mol<br />
var cAs = Number(cAsNode.value) * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = Number(McelldrywetNode.value); // kg/kg<br />
var rhocell = Number(rhocellNode.value); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(AspercellvolumeNode, Aspercellvolume);<br />
setOutput(molAspercellvolumeNode, molAspercellvolume);<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|thumb|In addition to binding to As(III), ArsR can repress expression of OpG. This is a negative feedback to the operon. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen1997]]).<br />
In the <i>E. coli</i> top10 there is only OpG present on the genome, which produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD (not used in this project). We intend to introduce instead OpH, which constitutively produces ArsR, in order to produce an abundance of ArsR.<br />
]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. {{todo|TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?}}<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
K1<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
K3<sub>d</sub> (ArsR<sub>opn</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;1 (ArsR) = <input type="text" id="tau1" value="30"/> min (??, dilution)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
&beta;1 (ArsR) = <input type="text" id="beta1" value="1000"/> 1/second (???)<br/><br />
&beta;3 (ArsR constitutive) = <input type="text" id="beta3" value="1000"/> 1/second (???)<br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
OpG<sub>total</sub> = <input type="text" id="OpGTotalPerCell" value="1"/> per cell (?)<br/><br />
OpH = <input type="text" id="OpHPerCell" value="10"/> per cell (??)<br/><br />
V<sub>cell</sub> = <input type="text" id="Vc" value="1"/> &micro;m<sup>3</sup> </html>[http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi]<html><br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>Unbound substances</dt><br />
<dd><br />
OpG / OpG<sub>total</sub> = <span id="OpGFraction"></span><br/><br />
ArsR = <span id="ArsRConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>Bound substances</dt><br />
<dd><br />
<!--ArsR<sub>As</sub> = <span id="ArsRAs3Concentration"></span> &micro;M<br/>--><br />
ArsR<sub>op</sub> = <span id="ArsROpConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="As3TotalConcentration"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="As3TotalFactor"></span><br/><br />
</dd><br />
</dl><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Input<br />
var K1dNode = document.getElementById("K1d");<br />
var K3dNode = document.getElementById("K3d");<br />
var tau1Node = document.getElementById("tau1");<br />
var beta1Node = document.getElementById("beta1");<br />
var beta3Node = document.getElementById("beta3");<br />
//var As3Node = document.getElementById("As3Concentration");<br />
var OpGTPerCellNode = document.getElementById("OpGTotalPerCell");<br />
var OpHPerCellNode = document.getElementById("OpHPerCell");<br />
var VcNode = document.getElementById("Vc");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var OpGFractionNode = document.getElementById("OpGFraction");<br />
var ArsRNode = document.getElementById("ArsRConcentration");<br />
//var ArsRAs3Node = document.getElementById("ArsRAs3Concentration");<br />
var ArsROpNode = document.getElementById("ArsROpConcentration");<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Outputs<br />
//var As3TNode = document.getElementById("As3TotalConcentration");<br />
var As3TFactorNode = document.getElementById("As3TotalFactor");<br />
<br />
// Read inputs<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var K1d = Number(K1dNode.value) * 1e-6; // micromolar -> molar<br />
var K3d2 = Math.pow(Number(K3dNode.value) * 1e-6,2); // micromolar -> molar<br />
var tau1 = Number(tau1Node.value) * 60; // minutes -> seconds<br />
var beta1 = Number(beta1Node.value); // 1/second<br />
var beta3 = Number(beta3Node.value); // 1/second<br />
//var As3 = Number(As3Node.value) * 1e-6; // micromolar -> molar<br />
var Vc = Number(VcNode.value) * 1e-15; // micrometer^3/cell -> liter/cell<br />
var OpGT= Number(OpGTPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
var OpH = Number(OpHPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
// Fixed point iteration<br />
var ArsR= 1;<br />
do {<br />
var fx = Math.pow(ArsR,3) - (tau1/Math.LN2)*beta3*OpH*Math.pow(ArsR,2) + K3d2*ArsR<br />
- K3d2*(tau1/Math.LN2)*(beta1*OpGT + beta3*OpH);<br />
var dfx = 3*Math.pow(ArsR,2) - 2*(tau1/Math.LN2)*beta3*OpH*ArsR + K3d2;<br />
var ddfx = 6*ArsR - 2*(tau1/Math.LN2)*beta3*OpH;<br />
ArsR = ArsR - 2*fx*dfx/(2*Math.pow(dfx,2)-fx*ddfx);<br />
} while(Math.abs(fx)>1e-6);<br />
<br />
var OpG = OpGT/(Math.pow(ArsR,2)/K3d2 + 1);<br />
//var ArsRAs3 = ArsR * As3 / K1d;<br />
var ArsROp = Math.pow(ArsR,2) * OpG / K3d2;<br />
<br />
var As3TFactor = 1 + ArsR/K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (OpGFractionNode) setOutput(OpGFractionNode, OpG/OpGT);<br />
if (ArsRNode) setOutput(ArsRNode, ArsR* 1e6);<br />
//if (ArsRAs3Node) setOutput(ArsRAs3Node, ArsRAs3* 1e6);<br />
if (ArsROpNode) setOutput(ArsROpNode, ArsROp* 1e6);<br />
<br />
// Set outputs<br />
//setOutput(As3TNode, As3Total* 1e6);<br />
setOutput(As3TFactorNode, As3TFactor);<br />
}<br />
</script><br />
</html><br />
<br />
In conclusion:<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors (they give a much higher increase in accumulation).<br />
* The model is not very sensitive to different values for K3d (with K3d=1M the accumulation factor is 7905.0 and with K3d=10<sup>-12</sup>M it is 7188.0).<br />
<br />
==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies ([[Team:Groningen/Literature#Lin2007-2|Lin2007]])<br />
**** Inductively coupled mass spectrometry (ICP-MS) (([[Team:Groningen/Literature#Meng2004|Meng2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen1997]]). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-09-09T12:30:11Z<p>Frans: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
==Introduction==<br />
<br />
Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..<br />
<br />
===Metallothioneins===<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) '''(Merrifield et al. 2004)'''. And they have readily been used to create cell based systems for purification of contaminated water [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Brady1994|Brady1994]]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one [[Team:Groningen/Literature#Chang1998|Chang1998]].<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium [[Team:Groningen/Literature#Deng2007|Deng2007]], arsenic [[Team:Groningen/Literature#Ngu2006|Ngu2006]], [[Team:Groningen/Literature#Kostal2004|Kostal2004]], [[Team:Groningen/Literature#Singh2008|Singh2008]], mercury [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Chen1998|Chen1997-2]], [[Team:Groningen/Literature#Deng2008|Deng2008]], nickel [[Team:Groningen/Literature#Deng2003|Deng2003]] or a combination of metals [[Team:Groningen/Literature#Chang1998|Chang1998]], [[Team:Groningen/Literature#Kao2008|Kao2008]].<br />
Metal-protein complexes can be quantified using a fluorescent molecule [[Team:Groningen/Literature#Cadosch2008 |Cadosch2008]].<br />
<br />
===Cloning strategy===<br />
{{todo}}[[Image:Cloning MT Transporter in AC3-new.pdf]]<br />
<br />
===Metals===<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) [[Team:Groningen/Literature#Ngu2006 |Ngu2006]] and fMT (the seaweed species ''fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity [[Team:Groningen/Literature#Singh2008 |Singh2008]], another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
ArsR is a trans-acting repressor that senses environmetal As(III)and regulates the chromosomal ars operon. The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified it's size corresponded to that of a homodimer, bound to promoter DNA. <br />
<br />
<br />
also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT wasn't succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
==Copper==<br />
pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) [[Team:Groningen/Literature#Gold2008 |Gold2008]]<br />
**Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).<br />
<br />
==Zinc==<br />
*Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) [[Team:Groningen/Literature#Blindauer2001|Blindauer2001]]<br />
<br />
===Alternatives===<br />
{{todo|Inclusion bodies}} [[Team:Groningen/Literature#Fowler1987 |Fowler1987]]<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
===Inhibitory characteristics?===<br />
<br />
==Modelling==<br />
===Arsenic - ArsR===<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Input<br />
var awAsNode = document.getElementById("awAs");<br />
var cAsNode = document.getElementById("cAs");<br />
var McelldrywetNode = document.getElementById("Mcelldrywet");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var AspercellvolumeNode = document.getElementById("Aspercellvolume");<br />
var molAspercellvolumeNode = document.getElementById("molAspercellvolume");<br />
<br />
// Read inputs<br />
var awAs = Number(awAsNode.value); // g/mol<br />
var cAs = Number(cAsNode.value) * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = Number(McelldrywetNode.value); // kg/kg<br />
var rhocell = Number(rhocellNode.value); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(AspercellvolumeNode, Aspercellvolume);<br />
setOutput(molAspercellvolumeNode, molAspercellvolume);<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|thumb|In addition to binding to As(III), ArsR can repress expression of OpG. This is a negative feedback to the operon. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen1997]]).<br />
In the <i>E. coli</i> top10 there is only OpG present on the genome, which produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD (not used in this project). We intend to introduce instead OpH, which constitutively produces ArsR, in order to produce an abundance of ArsR.<br />
]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. {{todo|TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?}}<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
K1<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
K3<sub>d</sub> (ArsR<sub>opn</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;1 (ArsR) = <input type="text" id="tau1" value="30"/> min (??, dilution)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
&beta;1 (ArsR) = <input type="text" id="beta1" value="1000"/> 1/second (???)<br/><br />
&beta;3 (ArsR constitutive) = <input type="text" id="beta3" value="1000"/> 1/second (???)<br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
OpG<sub>total</sub> = <input type="text" id="OpGTotalPerCell" value="1"/> per cell (?)<br/><br />
OpH = <input type="text" id="OpHPerCell" value="10"/> per cell (??)<br/><br />
V<sub>cell</sub> = <input type="text" id="Vc" value="1"/> &micro;m<sup>3</sup> </html>[http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi]<html><br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>Unbound substances</dt><br />
<dd><br />
OpG / OpG<sub>total</sub> = <span id="OpGFraction"></span><br/><br />
ArsR = <span id="ArsRConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>Bound substances</dt><br />
<dd><br />
<!--ArsR<sub>As</sub> = <span id="ArsRAs3Concentration"></span> &micro;M<br/>--><br />
ArsR<sub>op</sub> = <span id="ArsROpConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="As3TotalConcentration"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="As3TotalFactor"></span><br/><br />
</dd><br />
</dl><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Input<br />
var K1dNode = document.getElementById("K1d");<br />
var K3dNode = document.getElementById("K3d");<br />
var tau1Node = document.getElementById("tau1");<br />
var beta1Node = document.getElementById("beta1");<br />
var beta3Node = document.getElementById("beta3");<br />
//var As3Node = document.getElementById("As3Concentration");<br />
var OpGTPerCellNode = document.getElementById("OpGTotalPerCell");<br />
var OpHPerCellNode = document.getElementById("OpHPerCell");<br />
var VcNode = document.getElementById("Vc");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var OpGFractionNode = document.getElementById("OpGFraction");<br />
var ArsRNode = document.getElementById("ArsRConcentration");<br />
//var ArsRAs3Node = document.getElementById("ArsRAs3Concentration");<br />
var ArsROpNode = document.getElementById("ArsROpConcentration");<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Outputs<br />
//var As3TNode = document.getElementById("As3TotalConcentration");<br />
var As3TFactorNode = document.getElementById("As3TotalFactor");<br />
<br />
// Read inputs<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var K1d = Number(K1dNode.value) * 1e-6; // micromolar -> molar<br />
var K3d2 = Math.pow(Number(K3dNode.value) * 1e-6,2); // micromolar -> molar<br />
var tau1 = Number(tau1Node.value) * 60; // minutes -> seconds<br />
var beta1 = Number(beta1Node.value); // 1/second<br />
var beta3 = Number(beta3Node.value); // 1/second<br />
//var As3 = Number(As3Node.value) * 1e-6; // micromolar -> molar<br />
var Vc = Number(VcNode.value) * 1e-15; // micrometer^3/cell -> liter/cell<br />
var OpGT= Number(OpGTPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
var OpH = Number(OpHPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
// Fixed point iteration<br />
var ArsR= 1;<br />
do {<br />
var fx = Math.pow(ArsR,3) - (tau1/Math.LN2)*beta3*OpH*Math.pow(ArsR,2) + K3d2*ArsR<br />
- K3d2*(tau1/Math.LN2)*(beta1*OpGT + beta3*OpH);<br />
var dfx = 3*Math.pow(ArsR,2) - 2*(tau1/Math.LN2)*beta3*OpH*ArsR + K3d2;<br />
var ddfx = 6*ArsR - 2*(tau1/Math.LN2)*beta3*OpH;<br />
ArsR = ArsR - 2*fx*dfx/(2*Math.pow(dfx,2)-fx*ddfx);<br />
} while(Math.abs(fx)>1e-6);<br />
<br />
var OpG = OpGT/(Math.pow(ArsR,2)/K3d2 + 1);<br />
//var ArsRAs3 = ArsR * As3 / K1d;<br />
var ArsROp = Math.pow(ArsR,2) * OpG / K3d2;<br />
<br />
var As3TFactor = 1 + ArsR/K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (OpGFractionNode) setOutput(OpGFractionNode, OpG/OpGT);<br />
if (ArsRNode) setOutput(ArsRNode, ArsR* 1e6);<br />
//if (ArsRAs3Node) setOutput(ArsRAs3Node, ArsRAs3* 1e6);<br />
if (ArsROpNode) setOutput(ArsROpNode, ArsROp* 1e6);<br />
<br />
// Set outputs<br />
//setOutput(As3TNode, As3Total* 1e6);<br />
setOutput(As3TFactorNode, As3TFactor);<br />
}<br />
</script><br />
</html><br />
<br />
In conclusion:<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors (they give a much higher increase in accumulation).<br />
* The model is not very sensitive to different values for K3d (with K3d=1M the accumulation factor is 7905.0 and with K3d=10<sup>-12</sup>M it is 7188.0).<br />
<br />
==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies ([[Team:Groningen/Literature#Lin2007-2|Lin2007]])<br />
**** Inductively coupled mass spectrometry (ICP-MS) (([[Team:Groningen/Literature#Meng2004|Meng2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen1997]]). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest</div>Franshttp://2009.igem.org/Team:Groningen/Project/AccumulationTeam:Groningen/Project/Accumulation2009-09-09T10:14:23Z<p>Frans: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
==Introduction==<br />
<br />
Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..<br />
<br />
===Metallothioneins===<br />
Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) '''(Merrifield et al. 2004)'''. And they have readily been used to create cell based systems for purification of contaminated water [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Brady1994|Brady1994]]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one [[Team:Groningen/Literature#Chang1998|Chang1998]].<br />
Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium [[Team:Groningen/Literature#Deng2007|Deng2007]], arsenic [[Team:Groningen/Literature#Ngu2006|Ngu2006]], [[Team:Groningen/Literature#Kostal2004|Kostal2004]], [[Team:Groningen/Literature#Singh2008|Singh2008]], mercury [[Team:Groningen/Literature#Chen1998|Chen1998]], [[Team:Groningen/Literature#Chen1998|Chen1997-2]], [[Team:Groningen/Literature#Deng2008|Deng2008]], nickel [[Team:Groningen/Literature#Deng2003|Deng2003]] or a combination of metals [[Team:Groningen/Literature#Chang1998|Chang1998]], [[Team:Groningen/Literature#Kao2008|Kao2008]].<br />
Metal-protein complexes can be quantified using a fluorescent molecule [[Team:Groningen/Literature#Cadosch2008 |Cadosch2008]].<br />
<br />
===Cloning strategy===<br />
{{todo}}[[Image:Cloning MT Transporter in AC3-new.pdf]]<br />
<br />
===Metals===<br />
==Arsenic==<br />
For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) [[Team:Groningen/Literature#Ngu2006 |Ngu2006]] and fMT (the seaweed species ''fucus vesiculosis'') both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity [[Team:Groningen/Literature#Singh2008 |Singh2008]], another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature.<br />
ArsR is a trans-acting repressor that senses environmetal As(III). The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10<sup>-15</sup>M of AS(III) can induce the promotor. <br />
<br />
<br />
also see the [https://2009.igem.org/Team:Groningen/Project/Promoters|Metal sensitive promoters]. <br />
As ordering rh-MT wasn't succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.<br />
<br />
==Copper==<br />
pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) [[Team:Groningen/Literature#Gold2008 |Gold2008]]<br />
**Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).<br />
<br />
==Zinc==<br />
*Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) [[Team:Groningen/Literature#Blindauer2001|Blindauer2001]]<br />
<br />
===Alternatives===<br />
{{todo|Inclusion bodies}} [[Team:Groningen/Literature#Fowler1987 |Fowler1987]]<br><br />
{{todo|(Bacterio)Ferritins}}<br><br />
{{todo|Phytochelatins}}<br><br />
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]<br />
<br />
===Inhibitory characteristics?===<br />
<br />
==Modelling==<br />
===Arsenic - ArsR===<br />
Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).<br />
<br />
At this moment we use four different variables:<br />
<br />
# Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]<br />
# Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: [[Team:Groningen/Literature#Kostal2004|Kostal2004]]<br />
# The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]<br />
# Cell density. Source: see our [[Team:Groningen/Project/Vesicle|gas vesicle page]].<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
aw<sub>As(III)</sub> = <input type="text" id="awAs" value="74.92"/> g/mol<br/><br />
<nobr>n<sub>As(III)</sub> / M<sub>cell(dry)</sub> = <input type="text" id="cAs" value="2"/> millimole/kg</nobr><br/> <!-- Reasonable estimate --><br />
M<sub>cell(dry)</sub> / M<sub>cell(wet)</sub> = <input type="text" id="Mcelldrywet" value="0.3"/><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate --><br />
<br />
<button onClick="computeArsenicWeight()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicError" style="color:red"></div><br />
<nobr>As(III) intake per volume of cells</nobr><br/><br />
<nobr> = <span id="Aspercellvolume"></span> g/m<sup>3</sup></nobr><br/><br />
<nobr> = <span id="molAspercellvolume"></span> &micro;mol/liter (TODO: check)</nobr><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicWeight);<br />
<br />
function computeArsenicWeight() {<br />
// Input<br />
var awAsNode = document.getElementById("awAs");<br />
var cAsNode = document.getElementById("cAs");<br />
var McelldrywetNode = document.getElementById("Mcelldrywet");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var arsenicErrorNode = document.getElementById("arsenicError");<br />
arsenicErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var AspercellvolumeNode = document.getElementById("Aspercellvolume");<br />
var molAspercellvolumeNode = document.getElementById("molAspercellvolume");<br />
<br />
// Read inputs<br />
var awAs = Number(awAsNode.value); // g/mol<br />
var cAs = Number(cAsNode.value) * 1e-3; // mmol/kg -> mol/kg<br />
var Mcelldrywet = Number(McelldrywetNode.value); // kg/kg<br />
var rhocell = Number(rhocellNode.value); // kg/m^3<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var Aspercellvolume = awAs * cAs * Mcelldrywet * rhocell;<br />
var molAspercellvolume = cAs * Mcelldrywet * rhocell * 1e3;<br />
// 1e-3 to convert from /m^3 to /L and 1e6 to convert from mole to micromole<br />
} catch(err) {<br />
arsenicErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(AspercellvolumeNode, Aspercellvolume);<br />
setOutput(molAspercellvolumeNode, molAspercellvolume);<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
<br />
As per cell volume = awAs * nAs(III) /<br />
Mcell(dry) * Mcelldrywet * rhocell<br />
mol As per cell volume = nAs(III) / <br />
Mcell(dry) * Mcelldrywet * rhocell<br />
<br />
</pre><br />
|}<br />
<br />
[[Image:Arsenic_accumulation.png|thumb|In addition to binding to As(III), ArsR can repress expression of OpG. This is a negative feedback to the operon. In effect this regulates the production of ArsR based on the As(III) concentration ([[Team:Groningen/Literature#Chen1997|Chen1997]]).<br />
In the <i>E. coli</i> top10 there is only OpG present on the genome, which produce ArsR (see [[Team:Groningen/BLAST|BLAST]] results). There are plasmids which produce both ArsR and ArsD (not used in this project). We intend to introduce instead OpH, which constitutively produces ArsR, in order to produce an abundance of ArsR.<br />
]]<br />
<br />
At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.<br />
<br />
The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell.<br />
See our [[Team:Groningen/Modelling/Arsenic|Modelling]] page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. {{todo|TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?}}<br />
<br />
<html><br />
<table style="background:#efe;border:1px solid #9c9;padding:1em;"><tr><td><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Dissociation constants</dt><br />
<dd><br />
K1<sub>d</sub> (ArsR<sub>As</sub>) = <input type="text" id="K1d" value="6"/> &micro;M (??)<br/><br />
K3<sub>d</sub> (ArsR<sub>opn</sub>) = <input type="text" id="K3d" value="0.33"/> &micro;M (</html>[[Team:Groningen/Literature#Chen1997|Chen1997]]<html>)<br/><br />
</dd><br />
<dt>Half-lifes</dt><br />
<dd><br />
&tau;1 (ArsR) = <input type="text" id="tau1" value="30"/> min (??, dilution)<br/><br />
</dd><br />
<dt>Production rates of the promoters</dt><br />
<dd><br />
&beta;1 (ArsR) = <input type="text" id="beta1" value="1000"/> 1/second (???)<br/><br />
&beta;3 (ArsR constitutive) = <input type="text" id="beta3" value="1000"/> 1/second (???)<br/><br />
</dd><br />
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>--><br />
<dt>Promoter concentrations<dt><br />
<dd><br />
OpG<sub>total</sub> = <input type="text" id="OpGTotalPerCell" value="1"/> per cell (?)<br/><br />
OpH = <input type="text" id="OpHPerCell" value="10"/> per cell (??)<br/><br />
V<sub>cell</sub> = <input type="text" id="Vc" value="1"/> &micro;m<sup>3</sup> </html>[http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi]<html><br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeArsenicEquilibrium()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="arsenicEquilibriumError" style="color:red"></div><br />
<dl><br />
<dt>Unbound substances</dt><br />
<dd><br />
OpG / OpG<sub>total</sub> = <span id="OpGFraction"></span><br/><br />
ArsR = <span id="ArsRConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>Bound substances</dt><br />
<dd><br />
<!--ArsR<sub>As</sub> = <span id="ArsRAs3Concentration"></span> &micro;M<br/>--><br />
ArsR<sub>op</sub> = <span id="ArsROpConcentration"></span> &micro;M<br/><br />
</dd><br />
<dt>"Accumulation factor"</dt><br />
<dd><br />
<!--As(III)<sub>total</sub> = <span id="As3TotalConcentration"></span> &micro;M<br/>--><br />
As(III)<sub>total</sub>/As(III) = <span id="As3TotalFactor"></span><br/><br />
</dd><br />
</dl><br />
</td><br />
</tr></table><br />
</td></tr></table><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeArsenicEquilibrium);<br />
<br />
function computeArsenicEquilibrium() {<br />
// Input<br />
var K1dNode = document.getElementById("K1d");<br />
var K3dNode = document.getElementById("K3d");<br />
var tau1Node = document.getElementById("tau1");<br />
var beta1Node = document.getElementById("beta1");<br />
var beta3Node = document.getElementById("beta3");<br />
//var As3Node = document.getElementById("As3Concentration");<br />
var OpGTPerCellNode = document.getElementById("OpGTotalPerCell");<br />
var OpHPerCellNode = document.getElementById("OpHPerCell");<br />
var VcNode = document.getElementById("Vc");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var OpGFractionNode = document.getElementById("OpGFraction");<br />
var ArsRNode = document.getElementById("ArsRConcentration");<br />
//var ArsRAs3Node = document.getElementById("ArsRAs3Concentration");<br />
var ArsROpNode = document.getElementById("ArsROpConcentration");<br />
var errorNode = document.getElementById("arsenicEquilibriumError");<br />
errorNode.innerHTML = '';<br />
<br />
// Outputs<br />
//var As3TNode = document.getElementById("As3TotalConcentration");<br />
var As3TFactorNode = document.getElementById("As3TotalFactor");<br />
<br />
// Read inputs<br />
var avogadro = 6.02214179e23; // 1/mol<br />
var K1d = Number(K1dNode.value) * 1e-6; // micromolar -> molar<br />
var K3d2 = Math.pow(Number(K3dNode.value) * 1e-6,2); // micromolar -> molar<br />
var tau1 = Number(tau1Node.value) * 60; // minutes -> seconds<br />
var beta1 = Number(beta1Node.value); // 1/second<br />
var beta3 = Number(beta3Node.value); // 1/second<br />
//var As3 = Number(As3Node.value) * 1e-6; // micromolar -> molar<br />
var Vc = Number(VcNode.value) * 1e-15; // micrometer^3/cell -> liter/cell<br />
var OpGT= Number(OpGTPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
var OpH = Number(OpHPerCellNode.value) / (avogadro*Vc); // 1/cell -> mol/liter<br />
<br />
// Compute density(/-ies)<br />
try {<br />
// Fixed point iteration<br />
var ArsR= 1;<br />
do {<br />
var fx = Math.pow(ArsR,3) - (tau1/Math.LN2)*beta3*OpH*Math.pow(ArsR,2) + K3d2*ArsR<br />
- K3d2*(tau1/Math.LN2)*(beta1*OpGT + beta3*OpH);<br />
var dfx = 3*Math.pow(ArsR,2) - 2*(tau1/Math.LN2)*beta3*OpH*ArsR + K3d2;<br />
var ddfx = 6*ArsR - 2*(tau1/Math.LN2)*beta3*OpH;<br />
ArsR = ArsR - 2*fx*dfx/(2*Math.pow(dfx,2)-fx*ddfx);<br />
} while(Math.abs(fx)>1e-6);<br />
<br />
var OpG = OpGT/(Math.pow(ArsR,2)/K3d2 + 1);<br />
//var ArsRAs3 = ArsR * As3 / K1d;<br />
var ArsROp = Math.pow(ArsR,2) * OpG / K3d2;<br />
<br />
var As3TFactor = 1 + ArsR/K1d;<br />
} catch(err) {<br />
errorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (OpGFractionNode) setOutput(OpGFractionNode, OpG/OpGT);<br />
if (ArsRNode) setOutput(ArsRNode, ArsR* 1e6);<br />
//if (ArsRAs3Node) setOutput(ArsRAs3Node, ArsRAs3* 1e6);<br />
if (ArsROpNode) setOutput(ArsROpNode, ArsROp* 1e6);<br />
<br />
// Set outputs<br />
//setOutput(As3TNode, As3Total* 1e6);<br />
setOutput(As3TFactorNode, As3TFactor);<br />
}<br />
</script><br />
</html><br />
<br />
In conclusion:<br />
<br />
* Even at the accumulation levels of Koster <i>et al.</i> the amount of arsenic accumulated in <i>E. coli</i> is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m<sup>3</sup>).<br />
* If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors (they give a much higher increase in accumulation).<br />
* The model is not very sensitive to different values for K3d (with K3d=1M the accumulation factor is 7905.0 and with K3d=10<sup>-12</sup>M it is 7188.0).<br />
<br />
==Planning and requirements:==<br />
<br />
* '''Modelling'''<br />
** Speed<br />
** Metaliotheines concentration <br />
** How often does the ArsR sensitive operator/operon occur in our <i>E. coli</i>?<br />
* '''Lab'''<br />
** Measurements<br />
*** Transport Assays<br />
**** Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies ([[Team:Groningen/Literature#Lin2007-2|Lin2007]])<br />
**** Inductively coupled mass spectrometry (ICP-MS) (([[Team:Groningen/Literature#Meng2004|Meng2004]])<br />
*** Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.<br />
*** Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)<br />
**** It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in [[Team:Groningen/Literature#Chen1997|Chen1997]]). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).<br />
*** Production rate of ArsR?<br />
** Biobrick Bba_K129004<br />
** Rest</div>Franshttp://2009.igem.org/User:FransUser:Frans2009-09-08T12:45:37Z<p>Frans: </p>
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Hey Hello <br />
<br />
my name is Frans Bianchi and I'am a team member of the 2009 Igem team from Groningen working in the lab.<br />
Currently I'm doing a master's in Molecular Biology and Biotechnology. So far Igem has been great fun and a big challenge especially making up a project and designing the system. The work in the Lab is also very challenging, some of the cloning has been done many times to make it work. Luckily my hobby is sailing which Itry to do as much as possible in my spare time. I'm also competing in sailing races all over the country with my J22.<br />
I 'll hope you had fun checking out the rest of our Wiki.<br />
<br />
<BR>[[Image:P7120076.JPG|500px|Frans]]</div>Franshttp://2009.igem.org/File:P7120076.JPGFile:P7120076.JPG2009-09-08T12:40:00Z<p>Frans: Foto Frans</p>
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<div>Foto Frans</div>Franshttp://2009.igem.org/User:FransUser:Frans2009-09-08T12:36:07Z<p>Frans: </p>
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<div>{{Team:Groningen/Header}}<br />
<br />
Hey Hello <br />
<br />
my name is Frans Bianchi and I'am a team member of the 2009 Igem team from Groningen working in the lab.<br />
Currently I'm doing a master's in Molecular Biology and Biotechnology. So far Igem has been great fun and a big challenge especially making up a project and designing the system. The work in the Lab is also very challenging, some of the cloning has been done many times to make it work. Luckily my hobby is sailing which Itry to do as much as possible in my spare time. I'm also competing in sailing races all over the country with my J22.<br />
I 'll hope you had fun checking out the rest of our Wiki.</div>Franshttp://2009.igem.org/Team:GroningenTeam:Groningen2009-09-04T13:31:19Z<p>Frans: </p>
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<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
<br />
{|<br />
|style="vertical-align:top"|[[Image:LogoIGEMGroningen.jpg]]<br />
|Welcome to the Wiki of the iGEM Groningen team! We are an interdisciplinary team of [[Team:Groningen/Team|11 enthusiastic students]] from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can also follow us on [http://twitter.com/igemgroningen twitter]!<br />
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[[Team:Groningen/Project|Our project]] is creating buoyant, metal accumulating bacteria to filter (heavy) metals from water. To ensure we have at least some success we have split up our project in four independent subprojects:<br />
<br />
*[[Team:Groningen/Project/Transport|Metal transport]]<br />
*[[Team:Groningen/Project/Accumulation|Metal accumulation]]<br />
*[[Team:Groningen/Project/Vesicle|Gas Vesicle]] <br />
*[[Team:Groningen/Project/Promoters|Metal sensitive promoters]]<br />
<br />
And don't forget to have a look at our [[Team:Groningen/Project_Plan|project plan]] (and other [[:Category:Team:Groningen/Disciplines/Project_Management|related documentation]]) as it is being developed. Also our [[:Category:Team:Groningen/Roles/Modeller|modellers]] have made a survey of some of the [[Team:Groningen/Brainstorm/Modelling|modelling technologies]] that have been used, our [[:Category:Team:Groningen/Roles/Treasurer|financial geniuses]] are raising funds and our [[:Category:Team:Groningen/Roles/Implementer|lab specialists]] are reading all sorts of [[Team:Groningen/Literature|interesting stuff on synthetic biology]].<br />
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'''Note for our team members:''' Layout ideas can be proposed/discussed/tried out [[Team:Groningen/Layout_Ideas|here]] and ideas for future work written down at [[Team:Groningen/Future]].<br />
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<div>(''iGEM: You must have a team page, a project page, and a notebook page.'')<br />
{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
<br />
{|<br />
|style="vertical-align:top"|[[Image:LogoIGEMGroningen.jpg]]<br />
|Welcome to the Wiki of the iGEM Groningen team! We are an interdisciplinary team of [[Team:Groningen/Team|11 enthusiastic students]] from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can also follow us on [http://twitter.com/igemgroningen twitter]!<br />
<br />
[[Image:Groningen_team2009_Martini.jpg|200px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
[[Team:Groningen/Project|Our project]] is creating buoyant, metal accumulating bacteria to filter (heavy) metals from water. To ensure we have at least some success we have split up our project in four independent subprojects:<br />
<br />
*[[Team:Groningen/Project/Transport|Metal transport]]<br />
*[[Team:Groningen/Project/Accumulation|Metal accumulation]]<br />
*[[Team:Groningen/Project/Vesicle|Gas Vesicle]] <br />
*[[Team:Groningen/Project/Promoters|Metal sensitive promoters]]<br />
<br />
And don't forget to have a look at our [[Team:Groningen/Project_Plan|project plan]] (and other [[:Category:Team:Groningen/Disciplines/Project_Management|related documentation]]) as it is being developed. Also our [[:Category:Team:Groningen/Roles/Modeller|modellers]] have made a survey of some of the [[Team:Groningen/Brainstorm/Modelling|modelling technologies]] that have been used, our [[:Category:Team:Groningen/Roles/Treasurer|financial geniuses]] are raising funds and our [[:Category:Team:Groningen/Roles/Implementer|lab specialists]] are reading all sorts of [[Team:Groningen/Literature|interesting stuff on synthetic biology]].<br />
<br />
'''Note for our team members:''' Layout ideas can be proposed/discussed/tried out [[Team:Groningen/Layout_Ideas|here]] and ideas for future work written down at [[Team:Groningen/Future]].<br />
|}<br />
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<html><a id="csb" href="http://www.centreforsyntheticbiology.eu/"><img style="WIDTH: 500px; HEIGHT: 85px"src="https://static.igem.org/mediawiki/igem.org/b/b0/Groningen2008_RUG_CSB.png" /></a></html><BR><br />
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<div></div>Franshttp://2009.igem.org/Team:Groningen/jqueryTeam:Groningen/jquery2009-09-04T13:15:29Z<p>Frans: Removing all content from page</p>
<hr />
<div></div>Franshttp://2009.igem.org/Team:Groningen/jquerycluetipTeam:Groningen/jquerycluetip2009-09-04T13:14:38Z<p>Frans: Removing all content from page</p>
<hr />
<div></div>Franshttp://2009.igem.org/Team:Groningen/FransTeam:Groningen/Frans2009-09-04T13:11:00Z<p>Frans: </p>
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<div id=main><br />
<div id=leftspace><br />
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<div id=mainmenu><br />
<div id=menuitem > <a href="#"> Overall description </a> </div> <br />
<div id=menuitem > <a href="#"> Motivation </a> </div> <br />
<div id=menuitem > <a href="#"> Project positioning </a> </div> <br />
<div id=menuitem > <a href="#"> Research proposal </a> </div> <br />
<div id=menuitem > <a href="#"> Methodology </a> </div> <br />
<div id=menuitem > <a href="#"> Why we differ? </a> </div><br />
<div id=menuitem > <a href="#"> Deliverables </a> </div><br />
<div id=menuitem > <a href="#"> Glance at future </a> </div><br />
<div id=menuitem > <a href="#"> References </a> </div><br />
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<li><a href="./">Overall description</a></li><br />
<li><a href="./">Motivation</a></li><br />
<li><a href="./">Project positioning</a></li><br />
<li><a href="./">Research proposal</a></li><br />
<li><a href="./">Methodology</a></li><br />
<li><a href="./">Why we differ?</a></li><br />
<li><a href="./">Deliverables</a></li><br />
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<li class="dir">Biology<br />
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<br />
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<br />
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<li><a href="./">Overall list</a></li><br />
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<li><a href="./">Overall list</a></li> <br />
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<br />
<li><a href="./">Failed ideas</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Overall description</b></font><br />
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</html></div>Franshttp://2009.igem.org/Team:Groningen/FransTeam:Groningen/Frans2009-09-04T12:52:12Z<p>Frans: New page: {{:Team:Edinburgh/cluetipstyle}} {{:Team:Edinburgh/dropdowncss}} {{:Team:Edinburgh/helpercss}} {{:Team:Edinburgh/defaultcss}} {{:Team:Edinburgh/defaultadvancedcss}} {{:Team:Edinburgh/light...</p>
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<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain" class="dir">Project</a><br />
<ul><br />
<li class="dir">Introduction<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Overall description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Motivation</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Project positioning</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Research proposal</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Methodology</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Why we differ?</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Deliverables</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Glance at the future</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">References</a></li><br />
</ul><br />
</li><br />
<br />
<li class="dir">Biology<br />
<ul><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Project overview</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Overall design</a></li><br />
<li class="dir">Materials&Methods<br />
<ul><br />
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<li class="dir">Protocols<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Overall list</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Composite Biobricks</a></li> <br />
</ul><br />
</li><br />
<br />
<li class="dir">Primers<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Overall list</a></li> <br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">TNT Primers</a></li> <br />
</ul><br />
</li><br />
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</ul><br />
</li><br />
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<li class="dir">Biobricks<br />
<ul><br />
<li class="dir">Submitted parts</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">TNT</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Tr2</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">ompRC</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">ONR</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">YFP</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">NSSR</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Pnir</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">YeaR</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">lux</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">luxGFP</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">lumP</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Double mutants</a></li><br />
</ul><br />
</li><br />
<li class="dir">Characterization<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">TNT</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Tr2</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">ompRC</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">ONR</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">YFP</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">NSSR</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Pnir</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">YeaR</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">lux</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">luxGFP</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">lumP</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Double mutants</a></li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Failed ideas</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Conclusions</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">References</a></li><br />
</ul><br />
</li><br />
<br />
<li class="dir">Modelling<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Introduction</a></li><br />
<li class="dir">Gene regulatory network<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Assembly methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Lux operon</a></li><br />
<li class="dir">Appendix<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Kappa code</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">SBML code</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Rate values</a></li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
<li class="dir">Real life application<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Diffusion graphs</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Animation</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Coding</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Failed ideas</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Scale up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Possible commersalization</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/projectmain">Glance at the future</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<li class="dir">Real world application</li><br />
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</ul><br />
</li><br />
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<li class="dir">Synthetic biology & iGem related<br />
<ul><br />
<li class="dir">Ethics<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Religious perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Public perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Survey and conclusions</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Summary (synthetic bilogy pro and contras)</a></li><br />
</ul><br />
</li><br />
<br />
<li class="dir">Data trading<br />
<ul><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><a href="./" class="dir">Team</a><br />
<ul><br />
<li class="empty">Introduction</li><br />
<li><a href="./">Team introduction</a></li><br />
<li><a href="./">Edinburgh University</a></li><br />
<li><a href="./">Team members</a></li><br />
<li><a href="./">Advisors</a></li><br />
<li><a href="./">Supervisors</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="./">Gallery</a></li><br />
<li><a href="./">Sponsors</a></li><br />
<li><a href="./">Acknowledgements</a></li><br />
<li><a href="./">Contacts</a></li><br />
</ul><br />
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<b>iGEM PROJECT "TNT/RDX DETECTOR AND BIOREMEDIATOR" <a class="load-local" href="#loadme" rel="#loadme">[Captain Planet, he's our hero!]</a></b><br />
<br /><br /><br />
During our brainstorming we discussed a <a class="load-local" href="#loadme" rel="#loadme"><b>variety of topics</b></a> which we found stimulating and interesting. Topics ranged from <a class="load-local" href="#loadme" rel="#loadme"><b>employing bacteria to desalinate water</b></a>, to <a class="load-local" href="#loadme" rel="#loadme"><b>destroying deadly algal blooms</b></a>, and making a synthetic vesicle construct. The latter would enable direct targeting of substances to specific tissues; something that could hopefully bring researchers a step closer to discovering a less invasive cure for diseases such as cancer and HIV.<br />
<br /><br /><br />
All these ideas are very exciting and compelling, and even though they were so diverse they had something in common. If successful, they could change the public perception of Synthetic Biology from being a frivolous endeavour by mad scientists, to a discipline with real life applications.<br />
<br /><br /><br />
A global initiative to develop Synthetic Biology for practical uses can greatly improve the quality of life of people not only directly, but also indirectly through improving the environment we live in.<br />
<br /><br /><br />
Something else that these projects had in common was that we could not actually work on them. Reasons ranging from the lack of necessary equipment, a permit to work with mammalian cells, or modelling proved that the project was not industrially and economically feasible.<br />
<br /><br /><br />
Finally, we arrived at the ideal project! It was both feasible, within the time-scale of iGEM, and exciting! As ambitious as it may sound, we are going to engineer Escherichia coli that can detect landmines efficiently and safely by eliminating the risk of injury to military personnel and civilians alike. The bacteria will detect both TNT and nitrites (a by-product of explosive degradation), and produce different light outputs, depending on the stimulus. For more detailed information on our project please check this link.<br />
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<b>PROJECT RELATED SECTIONS</b><br />
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New page: /*! * jQuery JavaScript Library v1.3.3pre * http://jquery.com/ * * Copyright (c) 2009 John Resig * Dual licensed under the MIT and GPL licenses. * http://docs.jquery.com/License * ...</p>
<hr />
<div>/*!<br />
* jQuery JavaScript Library v1.3.3pre<br />
* http://jquery.com/<br />
*<br />
* Copyright (c) 2009 John Resig<br />
* Dual licensed under the MIT and GPL licenses.<br />
* http://docs.jquery.com/License<br />
*<br />
* Date: 2009-06-04 13:36:23 -0400 (Thu, 04 Jun 2009)<br />
* Revision: 6372<br />
*/<br />
(function(window, undefined){<br />
<br />
// Define a local copy of jQuery<br />
var jQuery = function( selector, context ) {<br />
// The jQuery object is actually just the init constructor 'enhanced'<br />
return arguments.length === 0 ?<br />
rootjQuery :<br />
new jQuery.fn.init( selector, context );<br />
},<br />
<br />
// Map over jQuery in case of overwrite<br />
_jQuery = window.jQuery,<br />
<br />
// Map over the $ in case of overwrite<br />
_$ = window.$,<br />
<br />
// A central reference to the root jQuery(document)<br />
rootjQuery,<br />
<br />
// A simple way to check for HTML strings or ID strings<br />
// (both of which we optimize for)<br />
quickExpr = /^[^<]*(<(.|\s)+>)[^>]*$|^#([\w-]+)$/,<br />
<br />
// Is it a simple selector<br />
isSimple = /^.[^:#\[\.,]*$/,<br />
<br />
// Keep a UserAgent string for use with jQuery.browser<br />
userAgent = navigator.userAgent.toLowerCase(),<br />
<br />
// Save a reference to the core toString method<br />
toString = Object.prototype.toString;<br />
<br />
// Expose jQuery to the global object<br />
window.jQuery = window.$ = jQuery;<br />
<br />
jQuery.fn = jQuery.prototype = {<br />
init: function( selector, context ) {<br />
var match, elem, ret;<br />
<br />
// Handle $(""), $(null), or $(undefined)<br />
if ( !selector ) {<br />
this.length = 0;<br />
return this;<br />
}<br />
<br />
// Handle $(DOMElement)<br />
if ( selector.nodeType ) {<br />
this[0] = selector;<br />
this.length = 1;<br />
this.context = selector;<br />
return this;<br />
}<br />
<br />
// Handle HTML strings<br />
if ( typeof selector === "string" ) {<br />
// Are we dealing with HTML string or an ID?<br />
match = quickExpr.exec( selector );<br />
<br />
// Verify a match, and that no context was specified for #id<br />
if ( match && (match[1] || !context) ) {<br />
<br />
// HANDLE: $(html) -> $(array)<br />
if ( match[1] ) {<br />
selector = jQuery.clean( [ match[1] ], context );<br />
<br />
// HANDLE: $("#id")<br />
} else {<br />
elem = document.getElementById( match[3] );<br />
<br />
// Handle the case where IE and Opera return items<br />
// by name instead of ID<br />
if ( elem && elem.id !== match[3] ) {<br />
return rootjQuery.find( selector );<br />
}<br />
<br />
// Otherwise, we inject the element directly into the jQuery object<br />
ret = jQuery( elem || null );<br />
ret.context = document;<br />
ret.selector = selector;<br />
return ret;<br />
}<br />
<br />
// HANDLE: $(expr, $(...))<br />
} else if ( !context || context.jquery ) {<br />
return (context || rootjQuery).find( selector );<br />
<br />
// HANDLE: $(expr, context)<br />
// (which is just equivalent to: $(context).find(expr)<br />
} else {<br />
return jQuery( context ).find( selector );<br />
}<br />
<br />
// HANDLE: $(function)<br />
// Shortcut for document ready<br />
} else if ( jQuery.isFunction( selector ) ) {<br />
return rootjQuery.ready( selector );<br />
}<br />
<br />
// Make sure that old selector state is passed along<br />
if ( selector.selector && selector.context ) {<br />
this.selector = selector.selector;<br />
this.context = selector.context;<br />
}<br />
<br />
return this.setArray(jQuery.isArray( selector ) ?<br />
selector :<br />
jQuery.makeArray(selector));<br />
},<br />
<br />
// Start with an empty selector<br />
selector: "",<br />
<br />
// The current version of jQuery being used<br />
jquery: "1.3.3pre",<br />
<br />
// The number of elements contained in the matched element set<br />
size: function() {<br />
return this.length;<br />
},<br />
<br />
// Get the Nth element in the matched element set OR<br />
// Get the whole matched element set as a clean array<br />
get: function( num ) {<br />
return num == null ?<br />
<br />
// Return a 'clean' array<br />
Array.prototype.slice.call( this ) :<br />
<br />
// Return just the object<br />
this[ num ];<br />
},<br />
<br />
// Take an array of elements and push it onto the stack<br />
// (returning the new matched element set)<br />
pushStack: function( elems, name, selector ) {<br />
// Build a new jQuery matched element set<br />
var ret = jQuery( elems || null );<br />
<br />
// Add the old object onto the stack (as a reference)<br />
ret.prevObject = this;<br />
<br />
ret.context = this.context;<br />
<br />
if ( name === "find" ) {<br />
ret.selector = this.selector + (this.selector ? " " : "") + selector;<br />
} else if ( name ) {<br />
ret.selector = this.selector + "." + name + "(" + selector + ")";<br />
}<br />
<br />
// Return the newly-formed element set<br />
return ret;<br />
},<br />
<br />
// Force the current matched set of elements to become<br />
// the specified array of elements (destroying the stack in the process)<br />
// You should use pushStack() in order to do this, but maintain the stack<br />
setArray: function( elems ) {<br />
// Resetting the length to 0, then using the native Array push<br />
// is a super-fast way to populate an object with array-like properties<br />
this.length = 0;<br />
Array.prototype.push.apply( this, elems );<br />
<br />
return this;<br />
},<br />
<br />
// Execute a callback for every element in the matched set.<br />
// (You can seed the arguments with an array of args, but this is<br />
// only used internally.)<br />
each: function( callback, args ) {<br />
return jQuery.each( this, callback, args );<br />
},<br />
<br />
// Determine the position of an element within<br />
// the matched set of elements<br />
index: function( elem ) {<br />
if ( !elem || typeof elem === "string" ) {<br />
return jQuery.inArray( this[0],<br />
// If it receives a string, the selector is used<br />
// If it receives nothing, the siblings are used<br />
elem ? jQuery( elem ) : this.parent().children() );<br />
}<br />
// Locate the position of the desired element<br />
return jQuery.inArray(<br />
// If it receives a jQuery object, the first element is used<br />
elem.jquery ? elem[0] : elem, this );<br />
},<br />
<br />
is: function( selector ) {<br />
return !!selector && jQuery.multiFilter( selector, this ).length > 0;<br />
},<br />
<br />
// For internal use only.<br />
// Behaves like an Array's method, not like a jQuery method.<br />
push: [].push,<br />
sort: [].sort,<br />
splice: [].splice<br />
};<br />
<br />
// Give the init function the jQuery prototype for later instantiation<br />
jQuery.fn.init.prototype = jQuery.fn;<br />
<br />
jQuery.extend = jQuery.fn.extend = function() {<br />
// copy reference to target object<br />
var target = arguments[0] || {}, i = 1, length = arguments.length, deep = false, options, name, src, copy;<br />
<br />
// Handle a deep copy situation<br />
if ( typeof target === "boolean" ) {<br />
deep = target;<br />
target = arguments[1] || {};<br />
// skip the boolean and the target<br />
i = 2;<br />
}<br />
<br />
// Handle case when target is a string or something (possible in deep copy)<br />
if ( typeof target !== "object" && !jQuery.isFunction(target) ) {<br />
target = {};<br />
}<br />
<br />
// extend jQuery itself if only one argument is passed<br />
if ( length === i ) {<br />
target = this;<br />
--i;<br />
}<br />
<br />
for ( ; i < length; i++ ) {<br />
// Only deal with non-null/undefined values<br />
if ( (options = arguments[ i ]) != null ) {<br />
// Extend the base object<br />
for ( name in options ) {<br />
src = target[ name ];<br />
copy = options[ name ];<br />
<br />
// Prevent never-ending loop<br />
if ( target === copy ) {<br />
continue;<br />
}<br />
<br />
// Recurse if we're merging object values<br />
if ( deep && copy && typeof copy === "object" && !copy.nodeType ) {<br />
target[ name ] = jQuery.extend( deep,<br />
// Never move original objects, clone them<br />
src || ( copy.length != null ? [ ] : { } ), copy );<br />
<br />
// Don't bring in undefined values<br />
} else if ( copy !== undefined ) {<br />
target[ name ] = copy;<br />
}<br />
}<br />
}<br />
}<br />
<br />
// Return the modified object<br />
return target;<br />
};<br />
<br />
jQuery.extend({<br />
noConflict: function( deep ) {<br />
window.$ = _$;<br />
<br />
if ( deep ) {<br />
window.jQuery = _jQuery;<br />
}<br />
<br />
return jQuery;<br />
},<br />
<br />
// See test/unit/core.js for details concerning isFunction.<br />
// Since version 1.3, DOM methods and functions like alert<br />
// aren't supported. They return false on IE (#2968).<br />
isFunction: function( obj ) {<br />
return toString.call(obj) === "[object Function]";<br />
},<br />
<br />
isArray: function( obj ) {<br />
return toString.call(obj) === "[object Array]";<br />
},<br />
<br />
// check if an element is in a (or is an) XML document<br />
isXMLDoc: function( elem ) {<br />
return elem.nodeType === 9 && elem.documentElement.nodeName !== "HTML" ||<br />
!!elem.ownerDocument && elem.ownerDocument.documentElement.nodeName !== "HTML";<br />
},<br />
<br />
// Evalulates a script in a global context<br />
globalEval: function( data ) {<br />
if ( data && /\S/.test(data) ) {<br />
// Inspired by code by Andrea Giammarchi<br />
// http://webreflection.blogspot.com/2007/08/global-scope-evaluation-and-dom.html<br />
var head = document.getElementsByTagName("head")[0] || document.documentElement,<br />
script = document.createElement("script");<br />
<br />
script.type = "text/javascript";<br />
if ( jQuery.support.scriptEval ) {<br />
script.appendChild( document.createTextNode( data ) );<br />
} else {<br />
script.text = data;<br />
}<br />
<br />
// Use insertBefore instead of appendChild to circumvent an IE6 bug.<br />
// This arises when a base node is used (#2709).<br />
head.insertBefore( script, head.firstChild );<br />
head.removeChild( script );<br />
}<br />
},<br />
<br />
nodeName: function( elem, name ) {<br />
return elem.nodeName && elem.nodeName.toUpperCase() === name.toUpperCase();<br />
},<br />
<br />
// args is for internal usage only<br />
each: function( object, callback, args ) {<br />
var name, i = 0, length = object.length;<br />
<br />
if ( args ) {<br />
if ( length === undefined ) {<br />
for ( name in object ) {<br />
if ( callback.apply( object[ name ], args ) === false ) {<br />
break;<br />
}<br />
}<br />
} else {<br />
for ( ; i < length; ) {<br />
if ( callback.apply( object[ i++ ], args ) === false ) {<br />
break;<br />
}<br />
}<br />
}<br />
<br />
// A special, fast, case for the most common use of each<br />
} else {<br />
if ( length === undefined ) {<br />
for ( name in object ) {<br />
if ( callback.call( object[ name ], name, object[ name ] ) === false ) {<br />
break;<br />
}<br />
}<br />
} else {<br />
for ( var value = object[0];<br />
i < length && callback.call( value, i, value ) !== false; value = object[++i] ) {}<br />
}<br />
}<br />
<br />
return object;<br />
},<br />
<br />
trim: function( text ) {<br />
return (text || "").replace( /^\s+|\s+$/g, "" );<br />
},<br />
<br />
makeArray: function( array ) {<br />
var ret = [], i;<br />
<br />
if ( array != null ) {<br />
i = array.length;<br />
<br />
// The window, strings (and functions) also have 'length'<br />
if ( i == null || typeof array === "string" || jQuery.isFunction(array) || array.setInterval ) {<br />
ret[0] = array;<br />
} else {<br />
while ( i ) {<br />
ret[--i] = array[i];<br />
}<br />
}<br />
}<br />
<br />
return ret;<br />
},<br />
<br />
inArray: function( elem, array ) {<br />
for ( var i = 0, length = array.length; i < length; i++ ) {<br />
if ( array[ i ] === elem ) {<br />
return i;<br />
}<br />
}<br />
<br />
return -1;<br />
},<br />
<br />
merge: function( first, second ) {<br />
// We have to loop this way because IE & Opera overwrite the length<br />
// expando of getElementsByTagName<br />
var i = 0, elem, pos = first.length;<br />
<br />
// Also, we need to make sure that the correct elements are being returned<br />
// (IE returns comment nodes in a '*' query)<br />
if ( !jQuery.support.getAll ) {<br />
while ( (elem = second[ i++ ]) != null ) {<br />
if ( elem.nodeType !== 8 ) {<br />
first[ pos++ ] = elem;<br />
}<br />
}<br />
<br />
} else {<br />
while ( (elem = second[ i++ ]) != null ) {<br />
first[ pos++ ] = elem;<br />
}<br />
}<br />
<br />
return first;<br />
},<br />
<br />
unique: function( array ) {<br />
var ret = [], done = {}, id;<br />
<br />
try {<br />
for ( var i = 0, length = array.length; i < length; i++ ) {<br />
id = jQuery.data( array[ i ] );<br />
<br />
if ( !done[ id ] ) {<br />
done[ id ] = true;<br />
ret.push( array[ i ] );<br />
}<br />
}<br />
} catch( e ) {<br />
ret = array;<br />
}<br />
<br />
return ret;<br />
},<br />
<br />
grep: function( elems, callback, inv ) {<br />
var ret = [];<br />
<br />
// Go through the array, only saving the items<br />
// that pass the validator function<br />
for ( var i = 0, length = elems.length; i < length; i++ ) {<br />
if ( !inv !== !callback( elems[ i ], i ) ) {<br />
ret.push( elems[ i ] );<br />
}<br />
}<br />
<br />
return ret;<br />
},<br />
<br />
map: function( elems, callback ) {<br />
var ret = [], value;<br />
<br />
// Go through the array, translating each of the items to their<br />
// new value (or values).<br />
for ( var i = 0, length = elems.length; i < length; i++ ) {<br />
value = callback( elems[ i ], i );<br />
<br />
if ( value != null ) {<br />
ret[ ret.length ] = value;<br />
}<br />
}<br />
<br />
return ret.concat.apply( [], ret );<br />
},<br />
<br />
// Use of jQuery.browser is deprecated.<br />
// It's included for backwards compatibility and plugins,<br />
// although they should work to migrate away.<br />
browser: {<br />
version: (userAgent.match( /.+(?:rv|it|ra|ie)[\/: ]([\d.]+)/ ) || [0,'0'])[1],<br />
safari: /webkit/.test( userAgent ),<br />
opera: /opera/.test( userAgent ),<br />
msie: /msie/.test( userAgent ) && !/opera/.test( userAgent ),<br />
mozilla: /mozilla/.test( userAgent ) && !/(compatible|webkit)/.test( userAgent )<br />
}<br />
});<br />
<br />
// All jQuery objects should point back to these<br />
rootjQuery = jQuery(document);<br />
<br />
function evalScript( i, elem ) {<br />
if ( elem.src ) {<br />
jQuery.ajax({<br />
url: elem.src,<br />
async: false,<br />
dataType: "script"<br />
});<br />
} else {<br />
jQuery.globalEval( elem.text || elem.textContent || elem.innerHTML || "" );<br />
}<br />
<br />
if ( elem.parentNode ) {<br />
elem.parentNode.removeChild( elem );<br />
}<br />
}<br />
<br />
function now() {<br />
return (new Date).getTime();<br />
}<br />
var expando = "jQuery" + now(), uuid = 0, windowData = {};<br />
<br />
jQuery.extend({<br />
cache: {},<br />
<br />
data: function( elem, name, data ) {<br />
elem = elem == window ?<br />
windowData :<br />
elem;<br />
<br />
var id = elem[ expando ];<br />
<br />
// Compute a unique ID for the element<br />
if ( !id )<br />
id = elem[ expando ] = ++uuid;<br />
<br />
// Only generate the data cache if we're<br />
// trying to access or manipulate it<br />
if ( name && !jQuery.cache[ id ] )<br />
jQuery.cache[ id ] = {};<br />
<br />
// Prevent overriding the named cache with undefined values<br />
if ( data !== undefined )<br />
jQuery.cache[ id ][ name ] = data;<br />
<br />
// Return the named cache data, or the ID for the element<br />
return name ?<br />
jQuery.cache[ id ][ name ] :<br />
id;<br />
},<br />
<br />
removeData: function( elem, name ) {<br />
elem = elem == window ?<br />
windowData :<br />
elem;<br />
<br />
var id = elem[ expando ];<br />
<br />
// If we want to remove a specific section of the element's data<br />
if ( name ) {<br />
if ( jQuery.cache[ id ] ) {<br />
// Remove the section of cache data<br />
delete jQuery.cache[ id ][ name ];<br />
<br />
// If we've removed all the data, remove the element's cache<br />
name = "";<br />
<br />
for ( name in jQuery.cache[ id ] )<br />
break;<br />
<br />
if ( !name )<br />
jQuery.removeData( elem );<br />
}<br />
<br />
// Otherwise, we want to remove all of the element's data<br />
} else {<br />
// Clean up the element expando<br />
try {<br />
delete elem[ expando ];<br />
} catch(e){<br />
// IE has trouble directly removing the expando<br />
// but it's ok with using removeAttribute<br />
if ( elem.removeAttribute )<br />
elem.removeAttribute( expando );<br />
}<br />
<br />
// Completely remove the data cache<br />
delete jQuery.cache[ id ];<br />
}<br />
},<br />
queue: function( elem, type, data ) {<br />
if ( elem ){<br />
<br />
type = (type || "fx") + "queue";<br />
<br />
var q = jQuery.data( elem, type );<br />
<br />
if ( !q || jQuery.isArray(data) )<br />
q = jQuery.data( elem, type, jQuery.makeArray(data) );<br />
else if( data )<br />
q.push( data );<br />
<br />
}<br />
return q;<br />
},<br />
<br />
dequeue: function( elem, type ){<br />
var queue = jQuery.queue( elem, type ),<br />
fn = queue.shift();<br />
<br />
if( !type || type === "fx" )<br />
fn = queue[0];<br />
<br />
if( fn !== undefined )<br />
fn.call(elem);<br />
}<br />
});<br />
<br />
jQuery.fn.extend({<br />
data: function( key, value ){<br />
var parts = key.split(".");<br />
parts[1] = parts[1] ? "." + parts[1] : "";<br />
<br />
if ( value === undefined ) {<br />
var data = this.triggerHandler("getData" + parts[1] + "!", [parts[0]]);<br />
<br />
if ( data === undefined && this.length )<br />
data = jQuery.data( this[0], key );<br />
<br />
return data === undefined && parts[1] ?<br />
this.data( parts[0] ) :<br />
data;<br />
} else<br />
return this.trigger("setData" + parts[1] + "!", [parts[0], value]).each(function(){<br />
jQuery.data( this, key, value );<br />
});<br />
},<br />
<br />
removeData: function( key ){<br />
return this.each(function(){<br />
jQuery.removeData( this, key );<br />
});<br />
},<br />
queue: function(type, data){<br />
if ( typeof type !== "string" ) {<br />
data = type;<br />
type = "fx";<br />
}<br />
<br />
if ( data === undefined )<br />
return jQuery.queue( this[0], type );<br />
<br />
return this.each(function(){<br />
var queue = jQuery.queue( this, type, data );<br />
<br />
if( type == "fx" && queue.length == 1 )<br />
queue[0].call(this);<br />
});<br />
},<br />
dequeue: function(type){<br />
return this.each(function(){<br />
jQuery.dequeue( this, type );<br />
});<br />
}<br />
});/*!<br />
* Sizzle CSS Selector Engine - v1.0<br />
* Copyright 2009, The Dojo Foundation<br />
* Released under the MIT, BSD, and GPL Licenses.<br />
* More information: http://sizzlejs.com/<br />
*/<br />
(function(){<br />
<br />
var chunker = /((?:\((?:\([^()]+\)|[^()]+)+\)|\[(?:\[[^[\]]*\]|['"][^'"]*['"]|[^[\]'"]+)+\]|\\.|[^ >+~,(\[\\]+)+|[>+~])(\s*,\s*)?/g,<br />
done = 0,<br />
toString = Object.prototype.toString,<br />
hasDuplicate = false;<br />
<br />
var Sizzle = function(selector, context, results, seed) {<br />
results = results || [];<br />
var origContext = context = context || document;<br />
<br />
if ( context.nodeType !== 1 && context.nodeType !== 9 ) {<br />
return [];<br />
}<br />
<br />
if ( !selector || typeof selector !== "string" ) {<br />
return results;<br />
}<br />
<br />
var parts = [], m, set, checkSet, check, mode, extra, prune = true, contextXML = isXML(context);<br />
<br />
// Reset the position of the chunker regexp (start from head)<br />
chunker.lastIndex = 0;<br />
<br />
while ( (m = chunker.exec(selector)) !== null ) {<br />
parts.push( m[1] );<br />
<br />
if ( m[2] ) {<br />
extra = RegExp.rightContext;<br />
break;<br />
}<br />
}<br />
<br />
if ( parts.length > 1 && origPOS.exec( selector ) ) {<br />
if ( parts.length === 2 && Expr.relative[ parts[0] ] ) {<br />
set = posProcess( parts[0] + parts[1], context );<br />
} else {<br />
set = Expr.relative[ parts[0] ] ?<br />
[ context ] :<br />
Sizzle( parts.shift(), context );<br />
<br />
while ( parts.length ) {<br />
selector = parts.shift();<br />
<br />
if ( Expr.relative[ selector ] )<br />
selector += parts.shift();<br />
<br />
set = posProcess( selector, set );<br />
}<br />
}<br />
} else {<br />
// Take a shortcut and set the context if the root selector is an ID<br />
// (but not if it'll be faster if the inner selector is an ID)<br />
if ( !seed && parts.length > 1 && context.nodeType === 9 && !contextXML &&<br />
Expr.match.ID.test(parts[0]) && !Expr.match.ID.test(parts[parts.length - 1]) ) {<br />
var ret = Sizzle.find( parts.shift(), context, contextXML );<br />
context = ret.expr ? Sizzle.filter( ret.expr, ret.set )[0] : ret.set[0];<br />
}<br />
<br />
if ( context ) {<br />
var ret = seed ?<br />
{ expr: parts.pop(), set: makeArray(seed) } :<br />
Sizzle.find( parts.pop(), parts.length === 1 && (parts[0] === "~" || parts[0] === "+") && context.parentNode ? context.parentNode : context, contextXML );<br />
set = ret.expr ? Sizzle.filter( ret.expr, ret.set ) : ret.set;<br />
<br />
if ( parts.length > 0 ) {<br />
checkSet = makeArray(set);<br />
} else {<br />
prune = false;<br />
}<br />
<br />
while ( parts.length ) {<br />
var cur = parts.pop(), pop = cur;<br />
<br />
if ( !Expr.relative[ cur ] ) {<br />
cur = "";<br />
} else {<br />
pop = parts.pop();<br />
}<br />
<br />
if ( pop == null ) {<br />
pop = context;<br />
}<br />
<br />
Expr.relative[ cur ]( checkSet, pop, contextXML );<br />
}<br />
} else {<br />
checkSet = parts = [];<br />
}<br />
}<br />
<br />
if ( !checkSet ) {<br />
checkSet = set;<br />
}<br />
<br />
if ( !checkSet ) {<br />
throw "Syntax error, unrecognized expression: " + (cur || selector);<br />
}<br />
<br />
if ( toString.call(checkSet) === "[object Array]" ) {<br />
if ( !prune ) {<br />
results.push.apply( results, checkSet );<br />
} else if ( context && context.nodeType === 1 ) {<br />
for ( var i = 0; checkSet[i] != null; i++ ) {<br />
if ( checkSet[i] && (checkSet[i] === true || checkSet[i].nodeType === 1 && contains(context, checkSet[i])) ) {<br />
results.push( set[i] );<br />
}<br />
}<br />
} else {<br />
for ( var i = 0; checkSet[i] != null; i++ ) {<br />
if ( checkSet[i] && checkSet[i].nodeType === 1 ) {<br />
results.push( set[i] );<br />
}<br />
}<br />
}<br />
} else {<br />
makeArray( checkSet, results );<br />
}<br />
<br />
if ( extra ) {<br />
Sizzle( extra, origContext, results, seed );<br />
Sizzle.uniqueSort( results );<br />
}<br />
<br />
return results;<br />
};<br />
<br />
Sizzle.uniqueSort = function(results){<br />
if ( sortOrder ) {<br />
hasDuplicate = false;<br />
results.sort(sortOrder);<br />
<br />
if ( hasDuplicate ) {<br />
for ( var i = 1; i < results.length; i++ ) {<br />
if ( results[i] === results[i-1] ) {<br />
results.splice(i--, 1);<br />
}<br />
}<br />
}<br />
}<br />
};<br />
<br />
Sizzle.matches = function(expr, set){<br />
return Sizzle(expr, null, null, set);<br />
};<br />
<br />
Sizzle.find = function(expr, context, isXML){<br />
var set, match;<br />
<br />
if ( !expr ) {<br />
return [];<br />
}<br />
<br />
for ( var i = 0, l = Expr.order.length; i < l; i++ ) {<br />
var type = Expr.order[i], match;<br />
<br />
if ( (match = Expr.match[ type ].exec( expr )) ) {<br />
var left = RegExp.leftContext;<br />
<br />
if ( left.substr( left.length - 1 ) !== "\\" ) {<br />
match[1] = (match[1] || "").replace(/\\/g, "");<br />
set = Expr.find[ type ]( match, context, isXML );<br />
if ( set != null ) {<br />
expr = expr.replace( Expr.match[ type ], "" );<br />
break;<br />
}<br />
}<br />
}<br />
}<br />
<br />
if ( !set ) {<br />
set = context.getElementsByTagName("*");<br />
}<br />
<br />
return {set: set, expr: expr};<br />
};<br />
<br />
Sizzle.filter = function(expr, set, inplace, not){<br />
var old = expr, result = [], curLoop = set, match, anyFound,<br />
isXMLFilter = set && set[0] && isXML(set[0]);<br />
<br />
while ( expr && set.length ) {<br />
for ( var type in Expr.filter ) {<br />
if ( (match = Expr.match[ type ].exec( expr )) != null ) {<br />
var filter = Expr.filter[ type ], found, item;<br />
anyFound = false;<br />
<br />
if ( curLoop == result ) {<br />
result = [];<br />
}<br />
<br />
if ( Expr.preFilter[ type ] ) {<br />
match = Expr.preFilter[ type ]( match, curLoop, inplace, result, not, isXMLFilter );<br />
<br />
if ( !match ) {<br />
anyFound = found = true;<br />
} else if ( match === true ) {<br />
continue;<br />
}<br />
}<br />
<br />
if ( match ) {<br />
for ( var i = 0; (item = curLoop[i]) != null; i++ ) {<br />
if ( item ) {<br />
found = filter( item, match, i, curLoop );<br />
var pass = not ^ !!found;<br />
<br />
if ( inplace && found != null ) {<br />
if ( pass ) {<br />
anyFound = true;<br />
} else {<br />
curLoop[i] = false;<br />
}<br />
} else if ( pass ) {<br />
result.push( item );<br />
anyFound = true;<br />
}<br />
}<br />
}<br />
}<br />
<br />
if ( found !== undefined ) {<br />
if ( !inplace ) {<br />
curLoop = result;<br />
}<br />
<br />
expr = expr.replace( Expr.match[ type ], "" );<br />
<br />
if ( !anyFound ) {<br />
return [];<br />
}<br />
<br />
break;<br />
}<br />
}<br />
}<br />
<br />
// Improper expression<br />
if ( expr == old ) {<br />
if ( anyFound == null ) {<br />
throw "Syntax error, unrecognized expression: " + expr;<br />
} else {<br />
break;<br />
}<br />
}<br />
<br />
old = expr;<br />
}<br />
<br />
return curLoop;<br />
};<br />
<br />
var Expr = Sizzle.selectors = {<br />
order: [ "ID", "NAME", "TAG" ],<br />
match: {<br />
ID: /#((?:[\w\u00c0-\uFFFF_-]|\\.)+)/,<br />
CLASS: /\.((?:[\w\u00c0-\uFFFF_-]|\\.)+)/,<br />
NAME: /\[name=['"]*((?:[\w\u00c0-\uFFFF_-]|\\.)+)['"]*\]/,<br />
ATTR: /\[\s*((?:[\w\u00c0-\uFFFF_-]|\\.)+)\s*(?:(\S?=)\s*(['"]*)(.*?)\3|)\s*\]/,<br />
TAG: /^((?:[\w\u00c0-\uFFFF\*_-]|\\.)+)/,<br />
CHILD: /:(only|nth|last|first)-child(?:\((even|odd|[\dn+-]*)\))?/,<br />
POS: /:(nth|eq|gt|lt|first|last|even|odd)(?:\((\d*)\))?(?=[^-]|$)/,<br />
PSEUDO: /:((?:[\w\u00c0-\uFFFF_-]|\\.)+)(?:\((['"]*)((?:\([^\)]+\)|[^\2\(\)]*)+)\2\))?/<br />
},<br />
attrMap: {<br />
"class": "className",<br />
"for": "htmlFor"<br />
},<br />
attrHandle: {<br />
href: function(elem){<br />
return elem.getAttribute("href");<br />
}<br />
},<br />
relative: {<br />
"+": function(checkSet, part, isXML){<br />
var isPartStr = typeof part === "string",<br />
isTag = isPartStr && !/\W/.test(part),<br />
isPartStrNotTag = isPartStr && !isTag;<br />
<br />
if ( isTag && !isXML ) {<br />
part = part.toUpperCase();<br />
}<br />
<br />
for ( var i = 0, l = checkSet.length, elem; i < l; i++ ) {<br />
if ( (elem = checkSet[i]) ) {<br />
while ( (elem = elem.previousSibling) && elem.nodeType !== 1 ) {}<br />
<br />
checkSet[i] = isPartStrNotTag || elem && elem.nodeName === part ?<br />
elem || false :<br />
elem === part;<br />
}<br />
}<br />
<br />
if ( isPartStrNotTag ) {<br />
Sizzle.filter( part, checkSet, true );<br />
}<br />
},<br />
">": function(checkSet, part, isXML){<br />
var isPartStr = typeof part === "string";<br />
<br />
if ( isPartStr && !/\W/.test(part) ) {<br />
part = isXML ? part : part.toUpperCase();<br />
<br />
for ( var i = 0, l = checkSet.length; i < l; i++ ) {<br />
var elem = checkSet[i];<br />
if ( elem ) {<br />
var parent = elem.parentNode;<br />
checkSet[i] = parent.nodeName === part ? parent : false;<br />
}<br />
}<br />
} else {<br />
for ( var i = 0, l = checkSet.length; i < l; i++ ) {<br />
var elem = checkSet[i];<br />
if ( elem ) {<br />
checkSet[i] = isPartStr ?<br />
elem.parentNode :<br />
elem.parentNode === part;<br />
}<br />
}<br />
<br />
if ( isPartStr ) {<br />
Sizzle.filter( part, checkSet, true );<br />
}<br />
}<br />
},<br />
"": function(checkSet, part, isXML){<br />
var doneName = done++, checkFn = dirCheck;<br />
<br />
if ( !part.match(/\W/) ) {<br />
var nodeCheck = part = isXML ? part : part.toUpperCase();<br />
checkFn = dirNodeCheck;<br />
}<br />
<br />
checkFn("parentNode", part, doneName, checkSet, nodeCheck, isXML);<br />
},<br />
"~": function(checkSet, part, isXML){<br />
var doneName = done++, checkFn = dirCheck;<br />
<br />
if ( typeof part === "string" && !part.match(/\W/) ) {<br />
var nodeCheck = part = isXML ? part : part.toUpperCase();<br />
checkFn = dirNodeCheck;<br />
}<br />
<br />
checkFn("previousSibling", part, doneName, checkSet, nodeCheck, isXML);<br />
}<br />
},<br />
find: {<br />
ID: function(match, context, isXML){<br />
if ( typeof context.getElementById !== "undefined" && !isXML ) {<br />
var m = context.getElementById(match[1]);<br />
return m ? [m] : [];<br />
}<br />
},<br />
NAME: function(match, context, isXML){<br />
if ( typeof context.getElementsByName !== "undefined" ) {<br />
var ret = [], results = context.getElementsByName(match[1]);<br />
<br />
for ( var i = 0, l = results.length; i < l; i++ ) {<br />
if ( results[i].getAttribute("name") === match[1] ) {<br />
ret.push( results[i] );<br />
}<br />
}<br />
<br />
return ret.length === 0 ? null : ret;<br />
}<br />
},<br />
TAG: function(match, context){<br />
return context.getElementsByTagName(match[1]);<br />
}<br />
},<br />
preFilter: {<br />
CLASS: function(match, curLoop, inplace, result, not, isXML){<br />
match = " " + match[1].replace(/\\/g, "") + " ";<br />
<br />
if ( isXML ) {<br />
return match;<br />
}<br />
<br />
for ( var i = 0, elem; (elem = curLoop[i]) != null; i++ ) {<br />
if ( elem ) {<br />
if ( not ^ (elem.className && (" " + elem.className + " ").indexOf(match) >= 0) ) {<br />
if ( !inplace )<br />
result.push( elem );<br />
} else if ( inplace ) {<br />
curLoop[i] = false;<br />
}<br />
}<br />
}<br />
<br />
return false;<br />
},<br />
ID: function(match){<br />
return match[1].replace(/\\/g, "");<br />
},<br />
TAG: function(match, curLoop){<br />
for ( var i = 0; curLoop[i] === false; i++ ){}<br />
return curLoop[i] && isXML(curLoop[i]) ? match[1] : match[1].toUpperCase();<br />
},<br />
CHILD: function(match){<br />
if ( match[1] == "nth" ) {<br />
// parse equations like 'even', 'odd', '5', '2n', '3n+2', '4n-1', '-n+6'<br />
var test = /(-?)(\d*)n((?:\+|-)?\d*)/.exec(<br />
match[2] == "even" && "2n" || match[2] == "odd" && "2n+1" ||<br />
!/\D/.test( match[2] ) && "0n+" + match[2] || match[2]);<br />
<br />
// calculate the numbers (first)n+(last) including if they are negative<br />
match[2] = (test[1] + (test[2] || 1)) - 0;<br />
match[3] = test[3] - 0;<br />
}<br />
<br />
// TODO: Move to normal caching system<br />
match[0] = done++;<br />
<br />
return match;<br />
},<br />
ATTR: function(match, curLoop, inplace, result, not, isXML){<br />
var name = match[1].replace(/\\/g, "");<br />
<br />
if ( !isXML && Expr.attrMap[name] ) {<br />
match[1] = Expr.attrMap[name];<br />
}<br />
<br />
if ( match[2] === "~=" ) {<br />
match[4] = " " + match[4] + " ";<br />
}<br />
<br />
return match;<br />
},<br />
PSEUDO: function(match, curLoop, inplace, result, not){<br />
if ( match[1] === "not" ) {<br />
// If we're dealing with a complex expression, or a simple one<br />
if ( match[3].match(chunker).length > 1 || /^\w/.test(match[3]) ) {<br />
match[3] = Sizzle(match[3], null, null, curLoop);<br />
} else {<br />
var ret = Sizzle.filter(match[3], curLoop, inplace, true ^ not);<br />
if ( !inplace ) {<br />
result.push.apply( result, ret );<br />
}<br />
return false;<br />
}<br />
} else if ( Expr.match.POS.test( match[0] ) || Expr.match.CHILD.test( match[0] ) ) {<br />
return true;<br />
}<br />
<br />
return match;<br />
},<br />
POS: function(match){<br />
match.unshift( true );<br />
return match;<br />
}<br />
},<br />
filters: {<br />
enabled: function(elem){<br />
return elem.disabled === false && elem.type !== "hidden";<br />
},<br />
disabled: function(elem){<br />
return elem.disabled === true;<br />
},<br />
checked: function(elem){<br />
return elem.checked === true;<br />
},<br />
selected: function(elem){<br />
// Accessing this property makes selected-by-default<br />
// options in Safari work properly<br />
elem.parentNode.selectedIndex;<br />
return elem.selected === true;<br />
},<br />
parent: function(elem){<br />
return !!elem.firstChild;<br />
},<br />
empty: function(elem){<br />
return !elem.firstChild;<br />
},<br />
has: function(elem, i, match){<br />
return !!Sizzle( match[3], elem ).length;<br />
},<br />
header: function(elem){<br />
return /h\d/i.test( elem.nodeName );<br />
},<br />
text: function(elem){<br />
return "text" === elem.type;<br />
},<br />
radio: function(elem){<br />
return "radio" === elem.type;<br />
},<br />
checkbox: function(elem){<br />
return "checkbox" === elem.type;<br />
},<br />
file: function(elem){<br />
return "file" === elem.type;<br />
},<br />
password: function(elem){<br />
return "password" === elem.type;<br />
},<br />
submit: function(elem){<br />
return "submit" === elem.type;<br />
},<br />
image: function(elem){<br />
return "image" === elem.type;<br />
},<br />
reset: function(elem){<br />
return "reset" === elem.type;<br />
},<br />
button: function(elem){<br />
return "button" === elem.type || elem.nodeName.toUpperCase() === "BUTTON";<br />
},<br />
input: function(elem){<br />
return /input|select|textarea|button/i.test(elem.nodeName);<br />
}<br />
},<br />
setFilters: {<br />
first: function(elem, i){<br />
return i === 0;<br />
},<br />
last: function(elem, i, match, array){<br />
return i === array.length - 1;<br />
},<br />
even: function(elem, i){<br />
return i % 2 === 0;<br />
},<br />
odd: function(elem, i){<br />
return i % 2 === 1;<br />
},<br />
lt: function(elem, i, match){<br />
return i < match[3] - 0;<br />
},<br />
gt: function(elem, i, match){<br />
return i > match[3] - 0;<br />
},<br />
nth: function(elem, i, match){<br />
return match[3] - 0 == i;<br />
},<br />
eq: function(elem, i, match){<br />
return match[3] - 0 == i;<br />
}<br />
},<br />
filter: {<br />
PSEUDO: function(elem, match, i, array){<br />
var name = match[1], filter = Expr.filters[ name ];<br />
<br />
if ( filter ) {<br />
return filter( elem, i, match, array );<br />
} else if ( name === "contains" ) {<br />
return (elem.textContent || elem.innerText || "").indexOf(match[3]) >= 0;<br />
} else if ( name === "not" ) {<br />
var not = match[3];<br />
<br />
for ( i = 0, l = not.length; i < l; i++ ) {<br />
if ( not[i] === elem ) {<br />
return false;<br />
}<br />
}<br />
<br />
return true;<br />
}<br />
},<br />
CHILD: function(elem, match){<br />
var type = match[1], node = elem;<br />
switch (type) {<br />
case 'only':<br />
case 'first':<br />
while ( (node = node.previousSibling) ) {<br />
if ( node.nodeType === 1 ) return false;<br />
}<br />
if ( type == 'first') return true;<br />
node = elem;<br />
case 'last':<br />
while ( (node = node.nextSibling) ) {<br />
if ( node.nodeType === 1 ) return false;<br />
}<br />
return true;<br />
case 'nth':<br />
var first = match[2], last = match[3];<br />
<br />
if ( first == 1 && last == 0 ) {<br />
return true;<br />
}<br />
<br />
var doneName = match[0],<br />
parent = elem.parentNode;<br />
<br />
if ( parent && (parent.sizcache !== doneName || !elem.nodeIndex) ) {<br />
var count = 0;<br />
for ( node = parent.firstChild; node; node = node.nextSibling ) {<br />
if ( node.nodeType === 1 ) {<br />
node.nodeIndex = ++count;<br />
}<br />
} <br />
parent.sizcache = doneName;<br />
}<br />
<br />
var diff = elem.nodeIndex - last;<br />
if ( first == 0 ) {<br />
return diff == 0;<br />
} else {<br />
return ( diff % first == 0 && diff / first >= 0 );<br />
}<br />
}<br />
},<br />
ID: function(elem, match){<br />
return elem.nodeType === 1 && elem.getAttribute("id") === match;<br />
},<br />
TAG: function(elem, match){<br />
return (match === "*" && elem.nodeType === 1) || elem.nodeName === match;<br />
},<br />
CLASS: function(elem, match){<br />
return (" " + (elem.className || elem.getAttribute("class")) + " ")<br />
.indexOf( match ) > -1;<br />
},<br />
ATTR: function(elem, match){<br />
var name = match[1],<br />
result = Expr.attrHandle[ name ] ?<br />
Expr.attrHandle[ name ]( elem ) :<br />
elem[ name ] != null ?<br />
elem[ name ] :<br />
elem.getAttribute( name ),<br />
value = result + "",<br />
type = match[2],<br />
check = match[4];<br />
<br />
return result == null ?<br />
type === "!=" :<br />
type === "=" ?<br />
value === check :<br />
type === "*=" ?<br />
value.indexOf(check) >= 0 :<br />
type === "~=" ?<br />
(" " + value + " ").indexOf(check) >= 0 :<br />
!check ?<br />
value && result !== false :<br />
type === "!=" ?<br />
value != check :<br />
type === "^=" ?<br />
value.indexOf(check) === 0 :<br />
type === "$=" ?<br />
value.substr(value.length - check.length) === check :<br />
type === "|=" ?<br />
value === check || value.substr(0, check.length + 1) === check + "-" :<br />
false;<br />
},<br />
POS: function(elem, match, i, array){<br />
var name = match[2], filter = Expr.setFilters[ name ];<br />
<br />
if ( filter ) {<br />
return filter( elem, i, match, array );<br />
}<br />
}<br />
}<br />
};<br />
<br />
var origPOS = Expr.match.POS;<br />
<br />
for ( var type in Expr.match ) {<br />
Expr.match[ type ] = new RegExp( Expr.match[ type ].source + /(?![^\[]*\])(?![^\(]*\))/.source );<br />
}<br />
<br />
var makeArray = function(array, results) {<br />
array = Array.prototype.slice.call( array );<br />
<br />
if ( results ) {<br />
results.push.apply( results, array );<br />
return results;<br />
}<br />
<br />
return array;<br />
};<br />
<br />
// Perform a simple check to determine if the browser is capable of<br />
// converting a NodeList to an array using builtin methods.<br />
try {<br />
Array.prototype.slice.call( document.documentElement.childNodes );<br />
<br />
// Provide a fallback method if it does not work<br />
} catch(e){<br />
makeArray = function(array, results) {<br />
var ret = results || [];<br />
<br />
if ( toString.call(array) === "[object Array]" ) {<br />
Array.prototype.push.apply( ret, array );<br />
} else {<br />
if ( typeof array.length === "number" ) {<br />
for ( var i = 0, l = array.length; i < l; i++ ) {<br />
ret.push( array[i] );<br />
}<br />
} else {<br />
for ( var i = 0; array[i]; i++ ) {<br />
ret.push( array[i] );<br />
}<br />
}<br />
}<br />
<br />
return ret;<br />
};<br />
}<br />
<br />
var sortOrder;<br />
<br />
if ( document.documentElement.compareDocumentPosition ) {<br />
sortOrder = function( a, b ) {<br />
var ret = a.compareDocumentPosition(b) & 4 ? -1 : a === b ? 0 : 1;<br />
if ( ret === 0 ) {<br />
hasDuplicate = true;<br />
}<br />
return ret;<br />
};<br />
} else if ( "sourceIndex" in document.documentElement ) {<br />
sortOrder = function( a, b ) {<br />
var ret = a.sourceIndex - b.sourceIndex;<br />
if ( ret === 0 ) {<br />
hasDuplicate = true;<br />
}<br />
return ret;<br />
};<br />
} else if ( document.createRange ) {<br />
sortOrder = function( a, b ) {<br />
var aRange = a.ownerDocument.createRange(), bRange = b.ownerDocument.createRange();<br />
aRange.selectNode(a);<br />
aRange.collapse(true);<br />
bRange.selectNode(b);<br />
bRange.collapse(true);<br />
var ret = aRange.compareBoundaryPoints(Range.START_TO_END, bRange);<br />
if ( ret === 0 ) {<br />
hasDuplicate = true;<br />
}<br />
return ret;<br />
};<br />
}<br />
<br />
// Check to see if the browser returns elements by name when<br />
// querying by getElementById (and provide a workaround)<br />
(function(){<br />
// We're going to inject a fake input element with a specified name<br />
var form = document.createElement("div"),<br />
id = "script" + (new Date).getTime();<br />
form.innerHTML = "<a name='" + id + "'/>";<br />
<br />
// Inject it into the root element, check its status, and remove it quickly<br />
var root = document.documentElement;<br />
root.insertBefore( form, root.firstChild );<br />
<br />
// The workaround has to do additional checks after a getElementById<br />
// Which slows things down for other browsers (hence the branching)<br />
if ( !!document.getElementById( id ) ) {<br />
Expr.find.ID = function(match, context, isXML){<br />
if ( typeof context.getElementById !== "undefined" && !isXML ) {<br />
var m = context.getElementById(match[1]);<br />
return m ? m.id === match[1] || typeof m.getAttributeNode !== "undefined" && m.getAttributeNode("id").nodeValue === match[1] ? [m] : undefined : [];<br />
}<br />
};<br />
<br />
Expr.filter.ID = function(elem, match){<br />
var node = typeof elem.getAttributeNode !== "undefined" && elem.getAttributeNode("id");<br />
return elem.nodeType === 1 && node && node.nodeValue === match;<br />
};<br />
}<br />
<br />
root.removeChild( form );<br />
root = form = null; // release memory in IE<br />
})();<br />
<br />
(function(){<br />
// Check to see if the browser returns only elements<br />
// when doing getElementsByTagName("*")<br />
<br />
// Create a fake element<br />
var div = document.createElement("div");<br />
div.appendChild( document.createComment("") );<br />
<br />
// Make sure no comments are found<br />
if ( div.getElementsByTagName("*").length > 0 ) {<br />
Expr.find.TAG = function(match, context){<br />
var results = context.getElementsByTagName(match[1]);<br />
<br />
// Filter out possible comments<br />
if ( match[1] === "*" ) {<br />
var tmp = [];<br />
<br />
for ( var i = 0; results[i]; i++ ) {<br />
if ( results[i].nodeType === 1 ) {<br />
tmp.push( results[i] );<br />
}<br />
}<br />
<br />
results = tmp;<br />
}<br />
<br />
return results;<br />
};<br />
}<br />
<br />
// Check to see if an attribute returns normalized href attributes<br />
div.innerHTML = "<a href='#'></a>";<br />
if ( div.firstChild && typeof div.firstChild.getAttribute !== "undefined" &&<br />
div.firstChild.getAttribute("href") !== "#" ) {<br />
Expr.attrHandle.href = function(elem){<br />
return elem.getAttribute("href", 2);<br />
};<br />
}<br />
<br />
div = null; // release memory in IE<br />
})();<br />
<br />
if ( document.querySelectorAll ) (function(){<br />
var oldSizzle = Sizzle, div = document.createElement("div");<br />
div.innerHTML = "<p class='TEST'></p>";<br />
<br />
// Safari can't handle uppercase or unicode characters when<br />
// in quirks mode.<br />
if ( div.querySelectorAll && div.querySelectorAll(".TEST").length === 0 ) {<br />
return;<br />
}<br />
<br />
Sizzle = function(query, context, extra, seed){<br />
context = context || document;<br />
<br />
// Only use querySelectorAll on non-XML documents<br />
// (ID selectors don't work in non-HTML documents)<br />
if ( !seed && context.nodeType === 9 && !isXML(context) ) {<br />
try {<br />
return makeArray( context.querySelectorAll(query), extra );<br />
} catch(e){}<br />
}<br />
<br />
return oldSizzle(query, context, extra, seed);<br />
};<br />
<br />
for ( var prop in oldSizzle ) {<br />
Sizzle[ prop ] = oldSizzle[ prop ];<br />
}<br />
<br />
div = null; // release memory in IE<br />
})();<br />
<br />
if ( document.getElementsByClassName && document.documentElement.getElementsByClassName ) (function(){<br />
var div = document.createElement("div");<br />
div.innerHTML = "<div class='test e'></div><div class='test'></div>";<br />
<br />
// Opera can't find a second classname (in 9.6)<br />
if ( div.getElementsByClassName("e").length === 0 )<br />
return;<br />
<br />
// Safari caches class attributes, doesn't catch changes (in 3.2)<br />
div.lastChild.className = "e";<br />
<br />
if ( div.getElementsByClassName("e").length === 1 )<br />
return;<br />
<br />
Expr.order.splice(1, 0, "CLASS");<br />
Expr.find.CLASS = function(match, context, isXML) {<br />
if ( typeof context.getElementsByClassName !== "undefined" && !isXML ) {<br />
return context.getElementsByClassName(match[1]);<br />
}<br />
};<br />
<br />
div = null; // release memory in IE<br />
})();<br />
<br />
function dirNodeCheck( dir, cur, doneName, checkSet, nodeCheck, isXML ) {<br />
var sibDir = dir == "previousSibling" && !isXML;<br />
for ( var i = 0, l = checkSet.length; i < l; i++ ) {<br />
var elem = checkSet[i];<br />
if ( elem ) {<br />
if ( sibDir && elem.nodeType === 1 ){<br />
elem.sizcache = doneName;<br />
elem.sizset = i;<br />
}<br />
elem = elem[dir];<br />
var match = false;<br />
<br />
while ( elem ) {<br />
if ( elem.sizcache === doneName ) {<br />
match = checkSet[elem.sizset];<br />
break;<br />
}<br />
<br />
if ( elem.nodeType === 1 && !isXML ){<br />
elem.sizcache = doneName;<br />
elem.sizset = i;<br />
}<br />
<br />
if ( elem.nodeName === cur ) {<br />
match = elem;<br />
break;<br />
}<br />
<br />
elem = elem[dir];<br />
}<br />
<br />
checkSet[i] = match;<br />
}<br />
}<br />
}<br />
<br />
function dirCheck( dir, cur, doneName, checkSet, nodeCheck, isXML ) {<br />
var sibDir = dir == "previousSibling" && !isXML;<br />
for ( var i = 0, l = checkSet.length; i < l; i++ ) {<br />
var elem = checkSet[i];<br />
if ( elem ) {<br />
if ( sibDir && elem.nodeType === 1 ) {<br />
elem.sizcache = doneName;<br />
elem.sizset = i;<br />
}<br />
elem = elem[dir];<br />
var match = false;<br />
<br />
while ( elem ) {<br />
if ( elem.sizcache === doneName ) {<br />
match = checkSet[elem.sizset];<br />
break;<br />
}<br />
<br />
if ( elem.nodeType === 1 ) {<br />
if ( !isXML ) {<br />
elem.sizcache = doneName;<br />
elem.sizset = i;<br />
}<br />
if ( typeof cur !== "string" ) {<br />
if ( elem === cur ) {<br />
match = true;<br />
break;<br />
}<br />
<br />
} else if ( Sizzle.filter( cur, [elem] ).length > 0 ) {<br />
match = elem;<br />
break;<br />
}<br />
}<br />
<br />
elem = elem[dir];<br />
}<br />
<br />
checkSet[i] = match;<br />
}<br />
}<br />
}<br />
<br />
var contains = document.compareDocumentPosition ? function(a, b){<br />
return a.compareDocumentPosition(b) & 16;<br />
} : function(a, b){<br />
return a !== b && (a.contains ? a.contains(b) : true);<br />
};<br />
<br />
var isXML = function(elem){<br />
return elem.nodeType === 9 && elem.documentElement.nodeName !== "HTML" ||<br />
!!elem.ownerDocument && elem.ownerDocument.documentElement.nodeName !== "HTML";<br />
};<br />
<br />
var posProcess = function(selector, context){<br />
var tmpSet = [], later = "", match,<br />
root = context.nodeType ? [context] : context;<br />
<br />
// Position selectors must be done after the filter<br />
// And so must :not(positional) so we move all PSEUDOs to the end<br />
while ( (match = Expr.match.PSEUDO.exec( selector )) ) {<br />
later += match[0];<br />
selector = selector.replace( Expr.match.PSEUDO, "" );<br />
}<br />
<br />
selector = Expr.relative[selector] ? selector + "*" : selector;<br />
<br />
for ( var i = 0, l = root.length; i < l; i++ ) {<br />
Sizzle( selector, root[i], tmpSet );<br />
}<br />
<br />
return Sizzle.filter( later, tmpSet );<br />
};<br />
<br />
// EXPOSE<br />
jQuery.find = Sizzle;<br />
jQuery.expr = Sizzle.selectors;<br />
jQuery.expr[":"] = jQuery.expr.filters;<br />
<br />
Sizzle.selectors.filters.hidden = function(elem){<br />
var width = elem.offsetWidth, height = elem.offsetHeight;<br />
return ( width === 0 && height === 0 ) ?<br />
true :<br />
( width !== 0 && height !== 0 ) ?<br />
false :<br />
!!( jQuery.curCSS(elem, "display") === "none" );<br />
};<br />
<br />
Sizzle.selectors.filters.visible = function(elem){<br />
var width = elem.offsetWidth, height = elem.offsetHeight;<br />
return ( width === 0 && height === 0 ) ?<br />
false :<br />
( width > 0 && height > 0 ) ?<br />
true :<br />
!!( jQuery.curCSS(elem, "display") !== "none" );<br />
};<br />
<br />
Sizzle.selectors.filters.animated = function(elem){<br />
return jQuery.grep(jQuery.timers, function(fn){<br />
return elem === fn.elem;<br />
}).length;<br />
};<br />
<br />
jQuery.filter = jQuery.multiFilter = function( expr, elems, not ) {<br />
if ( not ) {<br />
expr = ":not(" + expr + ")";<br />
}<br />
<br />
return Sizzle.matches(expr, elems);<br />
};<br />
<br />
jQuery.dir = function( elem, dir ){<br />
var matched = [], cur = elem[dir];<br />
while ( cur && cur != document ) {<br />
if ( cur.nodeType == 1 )<br />
matched.push( cur );<br />
cur = cur[dir];<br />
}<br />
return matched;<br />
};<br />
<br />
jQuery.nth = function(cur, result, dir, elem){<br />
result = result || 1;<br />
var num = 0;<br />
<br />
for ( ; cur; cur = cur[dir] )<br />
if ( cur.nodeType == 1 && ++num == result )<br />
break;<br />
<br />
return cur;<br />
};<br />
<br />
jQuery.sibling = function(n, elem){<br />
var r = [];<br />
<br />
for ( ; n; n = n.nextSibling ) {<br />
if ( n.nodeType == 1 && n != elem )<br />
r.push( n );<br />
}<br />
<br />
return r;<br />
};<br />
<br />
return;<br />
<br />
window.Sizzle = Sizzle;<br />
<br />
})();<br />
jQuery.fn.extend({<br />
find: function( selector ) {<br />
var ret = this.pushStack( "", "find", selector ), length = 0;<br />
<br />
for ( var i = 0, l = this.length; i < l; i++ ) {<br />
length = ret.length;<br />
jQuery.find( selector, this[i], ret );<br />
<br />
if ( i > 0 ) {<br />
// Make sure that the results are unique<br />
for ( var n = length; n < ret.length; n++ ) {<br />
for ( var r = 0; r < length; r++ ) {<br />
if ( ret[r] === ret[n] ) {<br />
ret.splice(n--, 1);<br />
break;<br />
}<br />
}<br />
}<br />
}<br />
}<br />
<br />
return ret;<br />
},<br />
<br />
filter: function( selector ) {<br />
return this.pushStack(<br />
jQuery.isFunction( selector ) &&<br />
jQuery.grep(this, function(elem, i){<br />
return selector.call( elem, i );<br />
}) ||<br />
<br />
jQuery.multiFilter( selector, jQuery.grep(this, function(elem){<br />
return elem.nodeType === 1;<br />
}) ), "filter", selector );<br />
},<br />
<br />
closest: function( selector ) {<br />
var pos = jQuery.expr.match.POS.test( selector ) ? jQuery(selector) : null,<br />
closer = 0;<br />
<br />
return this.map(function(){<br />
var cur = this;<br />
while ( cur && cur.ownerDocument ) {<br />
if ( pos ? pos.index(cur) > -1 : jQuery(cur).is(selector) ) {<br />
jQuery.data(cur, "closest", closer);<br />
return cur;<br />
}<br />
cur = cur.parentNode;<br />
closer++;<br />
}<br />
});<br />
},<br />
<br />
not: function( selector ) {<br />
if ( typeof selector === "string" )<br />
// test special case where just one selector is passed in<br />
if ( isSimple.test( selector ) )<br />
return this.pushStack( jQuery.multiFilter( selector, this, true ), "not", selector );<br />
else<br />
selector = jQuery.multiFilter( selector, this );<br />
<br />
var isArrayLike = selector.length && selector[selector.length - 1] !== undefined && !selector.nodeType;<br />
return this.filter(function() {<br />
return isArrayLike ? jQuery.inArray( this, selector ) < 0 : this != selector;<br />
});<br />
},<br />
<br />
add: function( selector ) {<br />
return this.pushStack( jQuery.unique( jQuery.merge(<br />
this.get(),<br />
typeof selector === "string" ?<br />
jQuery( selector ) :<br />
jQuery.makeArray( selector )<br />
)));<br />
},<br />
<br />
eq: function( i ) {<br />
return this.slice( i, +i + 1 );<br />
},<br />
<br />
slice: function() {<br />
return this.pushStack( Array.prototype.slice.apply( this, arguments ),<br />
"slice", Array.prototype.slice.call(arguments).join(",") );<br />
},<br />
<br />
map: function( callback ) {<br />
return this.pushStack( jQuery.map(this, function(elem, i){<br />
return callback.call( elem, i, elem );<br />
}));<br />
},<br />
<br />
andSelf: function() {<br />
return this.add( this.prevObject );<br />
},<br />
<br />
end: function() {<br />
return this.prevObject || jQuery(null);<br />
}<br />
});<br />
<br />
jQuery.each({<br />
parent: function(elem){return elem.parentNode;},<br />
parents: function(elem){return jQuery.dir(elem,"parentNode");},<br />
next: function(elem){return jQuery.nth(elem,2,"nextSibling");},<br />
prev: function(elem){return jQuery.nth(elem,2,"previousSibling");},<br />
nextAll: function(elem){return jQuery.dir(elem,"nextSibling");},<br />
prevAll: function(elem){return jQuery.dir(elem,"previousSibling");},<br />
siblings: function(elem){return jQuery.sibling(elem.parentNode.firstChild,elem);},<br />
children: function(elem){return jQuery.sibling(elem.firstChild);},<br />
contents: function(elem){return jQuery.nodeName(elem,"iframe")?elem.contentDocument||elem.contentWindow.document:jQuery.makeArray(elem.childNodes);}<br />
}, function(name, fn){<br />
jQuery.fn[ name ] = function( selector ) {<br />
var ret = jQuery.map( this, fn );<br />
<br />
if ( selector && typeof selector == "string" )<br />
ret = jQuery.multiFilter( selector, ret );<br />
<br />
return this.pushStack( jQuery.unique( ret ), name, selector );<br />
};<br />
});jQuery.fn.extend({<br />
attr: function( name, value ) {<br />
var options = name, isFunction = jQuery.isFunction( value );<br />
<br />
if ( typeof name === "string" ) {<br />
// Are we setting the attribute?<br />
if ( value === undefined ) {<br />
return this.length ?<br />
jQuery.attr( this[0], name ) :<br />
null;<br />
<br />
// Convert name, value params to options hash format<br />
} else {<br />
options = {};<br />
options[ name ] = value;<br />
}<br />
}<br />
<br />
// For each element...<br />
for ( var i = 0, l = this.length; i < l; i++ ) {<br />
var elem = this[i];<br />
<br />
// Set all the attributes<br />
for ( var prop in options ) {<br />
value = options[prop];<br />
<br />
if ( isFunction ) {<br />
value = value.call( elem, i );<br />
}<br />
<br />
jQuery.attr( elem, prop, value );<br />
}<br />
}<br />
<br />
return this;<br />
},<br />
<br />
hasClass: function( selector ) {<br />
return !!selector && this.is( "." + selector );<br />
},<br />
<br />
val: function( value ) {<br />
if ( value === undefined ) {<br />
var elem = this[0];<br />
<br />
if ( elem ) {<br />
if( jQuery.nodeName( elem, 'option' ) )<br />
return (elem.attributes.value || {}).specified ? elem.value : elem.text;<br />
<br />
// We need to handle select boxes special<br />
if ( jQuery.nodeName( elem, "select" ) ) {<br />
var index = elem.selectedIndex,<br />
values = [],<br />
options = elem.options,<br />
one = elem.type == "select-one";<br />
<br />
// Nothing was selected<br />
if ( index < 0 )<br />
return null;<br />
<br />
// Loop through all the selected options<br />
for ( var i = one ? index : 0, max = one ? index + 1 : options.length; i < max; i++ ) {<br />
var option = options[ i ];<br />
<br />
if ( option.selected ) {<br />
// Get the specifc value for the option<br />
value = jQuery(option).val();<br />
<br />
// We don't need an array for one selects<br />
if ( one )<br />
return value;<br />
<br />
// Multi-Selects return an array<br />
values.push( value );<br />
}<br />
}<br />
<br />
return values;<br />
}<br />
<br />
// Everything else, we just grab the value<br />
return (elem.value || "").replace(/\r/g, "");<br />
<br />
}<br />
<br />
return undefined;<br />
}<br />
<br />
if ( typeof value === "number" )<br />
value += '';<br />
<br />
return this.each(function(){<br />
if ( this.nodeType != 1 )<br />
return;<br />
<br />
if ( jQuery.isArray(value) && /radio|checkbox/.test( this.type ) )<br />
this.checked = (jQuery.inArray(this.value, value) >= 0 ||<br />
jQuery.inArray(this.name, value) >= 0);<br />
<br />
else if ( jQuery.nodeName( this, "select" ) ) {<br />
var values = jQuery.makeArray(value);<br />
<br />
jQuery( "option", this ).each(function(){<br />
this.selected = (jQuery.inArray( this.value, values ) >= 0 ||<br />
jQuery.inArray( this.text, values ) >= 0);<br />
});<br />
<br />
if ( !values.length )<br />
this.selectedIndex = -1;<br />
<br />
} else<br />
this.value = value;<br />
});<br />
}<br />
});<br />
<br />
jQuery.each({<br />
removeAttr: function( name ) {<br />
jQuery.attr( this, name, "" );<br />
if (this.nodeType == 1)<br />
this.removeAttribute( name );<br />
},<br />
<br />
addClass: function( classNames ) {<br />
jQuery.className.add( this, classNames );<br />
},<br />
<br />
removeClass: function( classNames ) {<br />
jQuery.className.remove( this, classNames );<br />
},<br />
<br />
toggleClass: function( classNames, state ) {<br />
var type = typeof classNames;<br />
if ( type === "string" ) {<br />
// toggle individual class names<br />
var isBool = typeof state === "boolean", className, i = 0,<br />
classNames = classNames.split( /\s+/ );<br />
while ( (className = classNames[ i++ ]) ) {<br />
// check each className given, space seperated list<br />
state = isBool ? state : !jQuery.className.has( this, className );<br />
jQuery.className[ state ? "add" : "remove" ]( this, className );<br />
}<br />
} else if ( type === "undefined" || type === "boolean" ) {<br />
if ( this.className ) {<br />
// store className if set<br />
jQuery.data( this, "__className__", this.className );<br />
}<br />
// toggle whole className<br />
this.className = this.className || classNames === false ? "" : jQuery.data( this, "__className__" ) || "";<br />
}<br />
}<br />
}, function(name, fn){<br />
jQuery.fn[ name ] = function(){<br />
return this.each( fn, arguments );<br />
};<br />
});<br />
<br />
jQuery.extend({<br />
className: {<br />
// internal only, use addClass("class")<br />
add: function( elem, classNames ) {<br />
jQuery.each((classNames || "").split(/\s+/), function(i, className){<br />
if ( elem.nodeType == 1 && !jQuery.className.has( elem.className, className ) )<br />
elem.className += (elem.className ? " " : "") + className;<br />
});<br />
},<br />
<br />
// internal only, use removeClass("class")<br />
remove: function( elem, classNames ) {<br />
if (elem.nodeType == 1)<br />
elem.className = classNames !== undefined ?<br />
jQuery.grep(elem.className.split(/\s+/), function(className){<br />
return !jQuery.className.has( classNames, className );<br />
}).join(" ") :<br />
"";<br />
},<br />
<br />
// internal only, use hasClass("class")<br />
has: function( elem, className ) {<br />
return elem && jQuery.inArray( className, (elem.className || elem).toString().split(/\s+/) ) > -1;<br />
}<br />
},<br />
<br />
attr: function( elem, name, value ) {<br />
// don't set attributes on text and comment nodes<br />
if (!elem || elem.nodeType == 3 || elem.nodeType == 8)<br />
return undefined;<br />
<br />
var notxml = !elem.tagName || !jQuery.isXMLDoc( elem ),<br />
// Whether we are setting (or getting)<br />
set = value !== undefined;<br />
<br />
// Try to normalize/fix the name<br />
name = notxml && jQuery.props[ name ] || name;<br />
<br />
// Only do all the following if this is a node (faster for style)<br />
if ( elem.tagName ) {<br />
<br />
// These attributes require special treatment<br />
var special = /href|src|style/.test( name );<br />
<br />
// Safari mis-reports the default selected property of a hidden option<br />
// Accessing the parent's selectedIndex property fixes it<br />
if ( name == "selected" && elem.parentNode )<br />
elem.parentNode.selectedIndex;<br />
<br />
// If applicable, access the attribute via the DOM 0 way<br />
if ( name in elem && notxml && !special ) {<br />
if ( set ){<br />
// We can't allow the type property to be changed (since it causes problems in IE)<br />
if ( name == "type" && elem.nodeName.match(/(button|input)/i) && elem.parentNode )<br />
throw "type property can't be changed";<br />
<br />
elem[ name ] = value;<br />
}<br />
<br />
// browsers index elements by id/name on forms, give priority to attributes.<br />
if( jQuery.nodeName( elem, "form" ) && elem.getAttributeNode(name) )<br />
return elem.getAttributeNode( name ).nodeValue;<br />
<br />
// elem.tabIndex doesn't always return the correct value when it hasn't been explicitly set<br />
// http://fluidproject.org/blog/2008/01/09/getting-setting-and-removing-tabindex-values-with-javascript/<br />
if ( name == "tabIndex" ) {<br />
var attributeNode = elem.getAttributeNode( "tabIndex" );<br />
return attributeNode && attributeNode.specified<br />
? attributeNode.value<br />
: elem.nodeName.match(/(button|input|object|select|textarea)/i)<br />
? 0<br />
: elem.nodeName.match(/^(a|area)$/i) && elem.href<br />
? 0<br />
: undefined;<br />
}<br />
<br />
return elem[ name ];<br />
}<br />
<br />
if ( !jQuery.support.style && notxml && name == "style" ) {<br />
if ( set )<br />
elem.style.cssText = "" + value;<br />
<br />
return elem.style.cssText;<br />
}<br />
<br />
if ( set )<br />
// convert the value to a string (all browsers do this but IE) see #1070<br />
elem.setAttribute( name, "" + value );<br />
<br />
var attr = !jQuery.support.hrefNormalized && notxml && special<br />
// Some attributes require a special call on IE<br />
? elem.getAttribute( name, 2 )<br />
: elem.getAttribute( name );<br />
<br />
// Non-existent attributes return null, we normalize to undefined<br />
return attr === null ? undefined : attr;<br />
}<br />
<br />
// elem is actually elem.style ... set the style<br />
// Using attr for specific style information is now deprecated. Use style insead.<br />
return jQuery.style(elem, name, value);<br />
}<br />
});jQuery.fn.extend({<br />
text: function( text ) {<br />
if ( typeof text !== "object" && text != null )<br />
return this.empty().append( (this[0] && this[0].ownerDocument || document).createTextNode( text ) );<br />
<br />
var ret = "";<br />
<br />
jQuery.each( text || this, function(){<br />
jQuery.each( this.childNodes, function(){<br />
if ( this.nodeType != 8 )<br />
ret += this.nodeType != 1 ?<br />
this.nodeValue :<br />
jQuery.fn.text( [ this ] );<br />
});<br />
});<br />
<br />
return ret;<br />
},<br />
<br />
wrapAll: function( html ) {<br />
if ( this[0] ) {<br />
// The elements to wrap the target around<br />
var wrap = jQuery( html, this[0].ownerDocument ).clone();<br />
<br />
if ( this[0].parentNode )<br />
wrap.insertBefore( this[0] );<br />
<br />
wrap.map(function(){<br />
var elem = this;<br />
<br />
while ( elem.firstChild )<br />
elem = elem.firstChild;<br />
<br />
return elem;<br />
}).append(this);<br />
}<br />
<br />
return this;<br />
},<br />
<br />
wrapInner: function( html ) {<br />
return this.each(function(){<br />
jQuery( this ).contents().wrapAll( html );<br />
});<br />
},<br />
<br />
wrap: function( html ) {<br />
return this.each(function(){<br />
jQuery( this ).wrapAll( html );<br />
});<br />
},<br />
<br />
append: function() {<br />
return this.domManip(arguments, true, function(elem){<br />
if (this.nodeType == 1)<br />
this.appendChild( elem );<br />
});<br />
},<br />
<br />
prepend: function() {<br />
return this.domManip(arguments, true, function(elem){<br />
if (this.nodeType == 1)<br />
this.insertBefore( elem, this.firstChild );<br />
});<br />
},<br />
<br />
before: function() {<br />
return this.domManip(arguments, false, function(elem){<br />
this.parentNode.insertBefore( elem, this );<br />
});<br />
},<br />
<br />
after: function() {<br />
return this.domManip(arguments, false, function(elem){<br />
this.parentNode.insertBefore( elem, this.nextSibling );<br />
});<br />
},<br />
<br />
clone: function( events ) {<br />
// Do the clone<br />
var ret = this.map(function(){<br />
if ( !jQuery.support.noCloneEvent && !jQuery.isXMLDoc(this) ) {<br />
// IE copies events bound via attachEvent when<br />
// using cloneNode. Calling detachEvent on the<br />
// clone will also remove the events from the orignal<br />
// In order to get around this, we use innerHTML.<br />
// Unfortunately, this means some modifications to<br />
// attributes in IE that are actually only stored<br />
// as properties will not be copied (such as the<br />
// the name attribute on an input).<br />
var html = this.outerHTML, ownerDocument = this.ownerDocument;<br />
if ( !html ) {<br />
var div = ownerDocument.createElement("div");<br />
div.appendChild( this.cloneNode(true) );<br />
html = div.innerHTML;<br />
}<br />
<br />
return jQuery.clean([html.replace(/ jQuery\d+="(?:\d+|null)"/g, "").replace(/^\s*/, "")], ownerDocument)[0];<br />
} else<br />
return this.cloneNode(true);<br />
});<br />
<br />
// Copy the events from the original to the clone<br />
if ( events === true ) {<br />
var orig = this.find("*").andSelf(), i = 0;<br />
<br />
ret.find("*").andSelf().each(function(){<br />
if ( this.nodeName !== orig[i].nodeName )<br />
return;<br />
<br />
var events = jQuery.data( orig[i], "events" );<br />
<br />
for ( var type in events ) {<br />
for ( var handler in events[ type ] ) {<br />
jQuery.event.add( this, type, events[ type ][ handler ], events[ type ][ handler ].data );<br />
}<br />
}<br />
<br />
i++;<br />
});<br />
}<br />
<br />
// Return the cloned set<br />
return ret;<br />
},<br />
<br />
html: function( value ) {<br />
return value === undefined ?<br />
(this[0] ?<br />
this[0].innerHTML.replace(/ jQuery\d+="(?:\d+|null)"/g, "") :<br />
null) :<br />
this.empty().append( value );<br />
},<br />
<br />
replaceWith: function( value ) {<br />
return this.after( value ).remove();<br />
},<br />
<br />
domManip: function( args, table, callback ) {<br />
if ( this[0] ) {<br />
var fragment = (this[0].ownerDocument || this[0]).createDocumentFragment(),<br />
scripts = jQuery.clean( args, (this[0].ownerDocument || this[0]), fragment ),<br />
first = fragment.firstChild;<br />
<br />
if ( first )<br />
for ( var i = 0, l = this.length; i < l; i++ )<br />
callback.call( root(this[i], first), this.length > 1 || i > 0 ?<br />
fragment.cloneNode(true) : fragment );<br />
<br />
if ( scripts )<br />
jQuery.each( scripts, evalScript );<br />
}<br />
<br />
return this;<br />
<br />
function root( elem, cur ) {<br />
return table && jQuery.nodeName(elem, "table") && jQuery.nodeName(cur, "tr") ?<br />
(elem.getElementsByTagName("tbody")[0] ||<br />
elem.appendChild(elem.ownerDocument.createElement("tbody"))) :<br />
elem;<br />
}<br />
}<br />
});<br />
<br />
jQuery.each({<br />
appendTo: "append",<br />
prependTo: "prepend",<br />
insertBefore: "before",<br />
insertAfter: "after",<br />
replaceAll: "replaceWith"<br />
}, function(name, original){<br />
jQuery.fn[ name ] = function( selector ) {<br />
var ret = [], insert = jQuery( selector );<br />
<br />
for ( var i = 0, l = insert.length; i < l; i++ ) {<br />
var elems = (i > 0 ? this.clone(true) : this).get();<br />
jQuery.fn[ original ].apply( jQuery(insert[i]), elems );<br />
ret = ret.concat( elems );<br />
}<br />
<br />
return this.pushStack( ret, name, selector );<br />
};<br />
});<br />
<br />
jQuery.each({<br />
remove: function( selector ) {<br />
if ( !selector || jQuery.multiFilter( selector, [ this ] ).length ) {<br />
if ( this.nodeType === 1 ) {<br />
cleanData( jQuery("*", this).add(this) );<br />
}<br />
<br />
if ( this.parentNode ) {<br />
this.parentNode.removeChild( this );<br />
}<br />
}<br />
},<br />
<br />
empty: function() {<br />
// Remove element nodes and prevent memory leaks<br />
if ( this.nodeType === 1 ) {<br />
cleanData( jQuery("*", this) );<br />
}<br />
<br />
// Remove any remaining nodes<br />
while ( this.firstChild ) {<br />
this.removeChild( this.firstChild );<br />
}<br />
}<br />
}, function(name, fn){<br />
jQuery.fn[ name ] = function(){<br />
return this.each( fn, arguments );<br />
};<br />
});<br />
<br />
jQuery.extend({<br />
clean: function( elems, context, fragment ) {<br />
context = context || document;<br />
<br />
// !context.createElement fails in IE with an error but returns typeof 'object'<br />
if ( typeof context.createElement === "undefined" )<br />
context = context.ownerDocument || context[0] && context[0].ownerDocument || document;<br />
<br />
// If a single string is passed in and it's a single tag<br />
// just do a createElement and skip the rest<br />
if ( !fragment && elems.length === 1 && typeof elems[0] === "string" ) {<br />
var match = /^<(\w+)\s*\/?>$/.exec(elems[0]);<br />
if ( match )<br />
return [ context.createElement( match[1] ) ];<br />
}<br />
<br />
var ret = [], scripts = [], div = context.createElement("div");<br />
<br />
jQuery.each(elems, function(i, elem){<br />
if ( typeof elem === "number" )<br />
elem += '';<br />
<br />
if ( !elem )<br />
return;<br />
<br />
// Convert html string into DOM nodes<br />
if ( typeof elem === "string" ) {<br />
// Fix "XHTML"-style tags in all browsers<br />
elem = elem.replace(/(<(\w+)[^>]*?)\/>/g, function(all, front, tag){<br />
return tag.match(/^(abbr|br|col|img|input|link|meta|param|hr|area|embed)$/i) ?<br />
all :<br />
front + "></" + tag + ">";<br />
});<br />
<br />
// Trim whitespace, otherwise indexOf won't work as expected<br />
var tags = elem.replace(/^\s+/, "").substring(0, 10).toLowerCase();<br />
<br />
var wrap =<br />
// option or optgroup<br />
!tags.indexOf("<opt") &&<br />
[ 1, "<select multiple='multiple'>", "</select>" ] ||<br />
<br />
!tags.indexOf("<leg") &&<br />
[ 1, "<fieldset>", "</fieldset>" ] ||<br />
<br />
tags.match(/^<(thead|tbody|tfoot|colg|cap)/) &&<br />
[ 1, "<table>", "</table>" ] ||<br />
<br />
!tags.indexOf("<tr") &&<br />
[ 2, "<table><tbody>", "</tbody></table>" ] ||<br />
<br />
// <thead> matched above<br />
(!tags.indexOf("<td") || !tags.indexOf("<th")) &&<br />
[ 3, "<table><tbody><tr>", "</tr></tbody></table>" ] ||<br />
<br />
!tags.indexOf("<col") &&<br />
[ 2, "<table><tbody></tbody><colgroup>", "</colgroup></table>" ] ||<br />
<br />
// IE can't serialize <link> and <script> tags normally<br />
!jQuery.support.htmlSerialize &&<br />
[ 1, "div<div>", "</div>" ] ||<br />
<br />
[ 0, "", "" ];<br />
<br />
// Go to html and back, then peel off extra wrappers<br />
div.innerHTML = wrap[1] + elem + wrap[2];<br />
<br />
// Move to the right depth<br />
while ( wrap[0]-- )<br />
div = div.lastChild;<br />
<br />
// Remove IE's autoinserted <tbody> from table fragments<br />
if ( !jQuery.support.tbody ) {<br />
<br />
// String was a <table>, *may* have spurious <tbody><br />
var hasBody = /<tbody/i.test(elem),<br />
tbody = !tags.indexOf("<table") && !hasBody ?<br />
div.firstChild && div.firstChild.childNodes :<br />
<br />
// String was a bare <thead> or <tfoot><br />
wrap[1] == "<table>" && !hasBody ?<br />
div.childNodes :<br />
[];<br />
<br />
for ( var j = tbody.length - 1; j >= 0 ; --j )<br />
if ( jQuery.nodeName( tbody[ j ], "tbody" ) && !tbody[ j ].childNodes.length )<br />
tbody[ j ].parentNode.removeChild( tbody[ j ] );<br />
<br />
}<br />
<br />
// IE completely kills leading whitespace when innerHTML is used<br />
if ( !jQuery.support.leadingWhitespace && /^\s/.test( elem ) )<br />
div.insertBefore( context.createTextNode( elem.match(/^\s*/)[0] ), div.firstChild );<br />
<br />
elem = jQuery.makeArray( div.childNodes );<br />
}<br />
<br />
if ( elem.nodeType )<br />
ret.push( elem );<br />
else<br />
ret = jQuery.merge( ret, elem );<br />
<br />
});<br />
<br />
if ( fragment ) {<br />
for ( var i = 0; ret[i]; i++ ) {<br />
if ( jQuery.nodeName( ret[i], "script" ) && (!ret[i].type || ret[i].type.toLowerCase() === "text/javascript") ) {<br />
scripts.push( ret[i].parentNode ? ret[i].parentNode.removeChild( ret[i] ) : ret[i] );<br />
} else {<br />
if ( ret[i].nodeType === 1 )<br />
ret.splice.apply( ret, [i + 1, 0].concat(jQuery.makeArray(ret[i].getElementsByTagName("script"))) );<br />
fragment.appendChild( ret[i] );<br />
}<br />
}<br />
<br />
return scripts;<br />
}<br />
<br />
return ret;<br />
}<br />
});<br />
<br />
function cleanData( elems ) {<br />
for ( var i = 0, l = elems.length; i < l; i++ ) {<br />
var id = elems[i][expando];<br />
if ( id ) {<br />
delete jQuery.cache[ id ];<br />
}<br />
}<br />
}<br />
/*<br />
* A number of helper functions used for managing events.<br />
* Many of the ideas behind this code originated from<br />
* Dean Edwards' addEvent library.<br />
*/<br />
jQuery.event = {<br />
<br />
// Bind an event to an element<br />
// Original by Dean Edwards<br />
add: function( elem, types, handler, data ) {<br />
if ( elem.nodeType === 3 || elem.nodeType === 8 ) {<br />
return;<br />
}<br />
<br />
// For whatever reason, IE has trouble passing the window object<br />
// around, causing it to be cloned in the process<br />
if ( elem.setInterval && ( elem !== window && !elem.frameElement ) ) {<br />
elem = window;<br />
}<br />
<br />
// Make sure that the function being executed has a unique ID<br />
if ( !handler.guid ) {<br />
handler.guid = this.guid++;<br />
}<br />
<br />
// if data is passed, bind to handler<br />
if ( data !== undefined ) {<br />
// Create temporary function pointer to original handler<br />
var fn = handler;<br />
<br />
// Create unique handler function, wrapped around original handler<br />
handler = this.proxy( fn );<br />
<br />
// Store data in unique handler<br />
handler.data = data;<br />
}<br />
<br />
// Init the element's event structure<br />
var events = jQuery.data( elem, "events" ) || jQuery.data( elem, "events", {} ),<br />
handle = jQuery.data( elem, "handle" ) || jQuery.data( elem, "handle", function() {<br />
// Handle the second event of a trigger and when<br />
// an event is called after a page has unloaded<br />
return typeof jQuery !== "undefined" && !jQuery.event.triggered ?<br />
jQuery.event.handle.apply( arguments.callee.elem, arguments ) :<br />
undefined;<br />
});<br />
// Add elem as a property of the handle function<br />
// This is to prevent a memory leak with non-native<br />
// event in IE.<br />
handle.elem = elem;<br />
<br />
// Handle multiple events separated by a space<br />
// jQuery(...).bind("mouseover mouseout", fn);<br />
types = types.split( /\s+/ );<br />
var type, i=0;<br />
while ( (type = types[ i++ ]) ) {<br />
// Namespaced event handlers<br />
var namespaces = type.split(".");<br />
type = namespaces.shift();<br />
handler.type = namespaces.slice().sort().join(".");<br />
<br />
// Get the current list of functions bound to this event<br />
var handlers = events[ type ],<br />
special = this.special[ type ] || {};<br />
<br />
if ( special.add ) {<br />
var modifiedHandler = special.add.call( elem, handler, data, namespaces );<br />
if ( modifiedHandler && jQuery.isFunction( modifiedHandler ) ) {<br />
modifiedHandler.guid = modifiedHandler.guid || handler.guid;<br />
handler = modifiedHandler;<br />
}<br />
}<br />
<br />
// Init the event handler queue<br />
if ( !handlers ) {<br />
handlers = events[ type ] = {};<br />
<br />
// Check for a special event handler<br />
// Only use addEventListener/attachEvent if the special<br />
// events handler returns false<br />
if ( !special.setup || special.setup.call( elem, data, namespaces ) === false ) {<br />
// Bind the global event handler to the element<br />
if ( elem.addEventListener ) {<br />
elem.addEventListener( type, handle, false );<br />
} else if ( elem.attachEvent ) {<br />
elem.attachEvent( "on" + type, handle );<br />
}<br />
}<br />
}<br />
<br />
// Add the function to the element's handler list<br />
handlers[ handler.guid ] = handler;<br />
<br />
// Keep track of which events have been used, for global triggering<br />
this.global[ type ] = true;<br />
}<br />
<br />
// Nullify elem to prevent memory leaks in IE<br />
elem = null;<br />
},<br />
<br />
guid: 1,<br />
global: {},<br />
<br />
// Detach an event or set of events from an element<br />
remove: function( elem, types, handler ) {<br />
// don't do events on text and comment nodes<br />
if ( elem.nodeType === 3 || elem.nodeType === 8 ) {<br />
return;<br />
}<br />
<br />
var events = jQuery.data( elem, "events" ), ret, type;<br />
<br />
if ( events ) {<br />
// Unbind all events for the element<br />
if ( types === undefined || (typeof types === "string" && types.charAt(0) === ".") ) {<br />
for ( type in events ) {<br />
this.remove( elem, type + (types || "") );<br />
}<br />
} else {<br />
// types is actually an event object here<br />
if ( types.type ) {<br />
handler = types.handler;<br />
types = types.type;<br />
}<br />
<br />
// Handle multiple events seperated by a space<br />
// jQuery(...).unbind("mouseover mouseout", fn);<br />
types = types.split(/\s+/);<br />
var i = 0;<br />
while ( (type = types[ i++ ]) ) {<br />
// Namespaced event handlers<br />
var namespaces = type.split(".");<br />
type = namespaces.shift();<br />
var all = !namespaces.length,<br />
namespace = new RegExp("(^|\\.)" + namespaces.slice().sort().join(".*\\.") + "(\\.|$)"),<br />
special = this.special[ type ] || {};<br />
<br />
if ( events[ type ] ) {<br />
// remove the given handler for the given type<br />
if ( handler ) {<br />
delete events[ type ][ handler.guid ];<br />
<br />
// remove all handlers for the given type<br />
} else {<br />
for ( var handle in events[ type ] ) {<br />
// Handle the removal of namespaced events<br />
if ( all || namespace.test( events[ type ][ handle ].type ) ) {<br />
delete events[ type ][ handle ];<br />
}<br />
}<br />
}<br />
<br />
if ( special.remove ) {<br />
special.remove.call( elem, namespaces );<br />
}<br />
<br />
// remove generic event handler if no more handlers exist<br />
for ( ret in events[ type ] ) {<br />
break;<br />
}<br />
if ( !ret ) {<br />
if ( !special.teardown || special.teardown.call( elem, namespaces ) === false ) {<br />
if ( elem.removeEventListener ) {<br />
elem.removeEventListener( type, jQuery.data( elem, "handle" ), false );<br />
} else if ( elem.detachEvent ) {<br />
elem.detachEvent( "on" + type, jQuery.data( elem, "handle" ) );<br />
}<br />
}<br />
ret = null;<br />
delete events[ type ];<br />
}<br />
}<br />
}<br />
}<br />
<br />
// Remove the expando if it's no longer used<br />
for ( ret in events ) {<br />
break;<br />
}<br />
if ( !ret ) {<br />
var handle = jQuery.data( elem, "handle" );<br />
if ( handle ) {<br />
handle.elem = null;<br />
}<br />
jQuery.removeData( elem, "events" );<br />
jQuery.removeData( elem, "handle" );<br />
}<br />
}<br />
},<br />
<br />
// bubbling is internal<br />
trigger: function( event, data, elem /*, bubbling */ ) {<br />
// Event object or event type<br />
var type = event.type || event,<br />
bubbling = arguments[3];<br />
<br />
if ( !bubbling ) {<br />
event = typeof event === "object" ?<br />
// jQuery.Event object<br />
event[expando] ? event :<br />
// Object literal<br />
jQuery.extend( jQuery.Event(type), event ) :<br />
// Just the event type (string)<br />
jQuery.Event(type);<br />
<br />
if ( type.indexOf("!") >= 0 ) {<br />
event.type = type = type.slice(0, -1);<br />
event.exclusive = true;<br />
}<br />
<br />
// Handle a global trigger<br />
if ( !elem ) {<br />
// Don't bubble custom events when global (to avoid too much overhead)<br />
event.stopPropagation();<br />
// Only trigger if we've ever bound an event for it<br />
if ( this.global[ type ] ) {<br />
jQuery.each( jQuery.cache, function() {<br />
if ( this.events && this.events[type] ) {<br />
jQuery.event.trigger( event, data, this.handle.elem );<br />
}<br />
});<br />
}<br />
}<br />
<br />
// Handle triggering a single element<br />
<br />
// don't do events on text and comment nodes<br />
if ( !elem || elem.nodeType === 3 || elem.nodeType === 8 ) {<br />
return undefined;<br />
}<br />
<br />
// Clean up in case it is reused<br />
event.result = undefined;<br />
event.target = elem;<br />
<br />
// Clone the incoming data, if any<br />
data = jQuery.makeArray( data );<br />
data.unshift( event );<br />
}<br />
<br />
event.currentTarget = elem;<br />
<br />
// Trigger the event, it is assumed that "handle" is a function<br />
var handle = jQuery.data( elem, "handle" );<br />
if ( handle ) {<br />
handle.apply( elem, data );<br />
}<br />
<br />
// Handle triggering native .onfoo handlers (and on links since we don't call .click() for links)<br />
if ( (!elem[ type ] || (jQuery.nodeName(elem, 'a') && type === "click")) && elem["on"+type] && elem["on"+type].apply( elem, data ) === false ) {<br />
event.result = false;<br />
}<br />
<br />
// Trigger the native events (except for clicks on links)<br />
if ( !bubbling && elem[ type ] && !event.isDefaultPrevented() && !(jQuery.nodeName(elem, 'a') && type === "click") ) {<br />
this.triggered = true;<br />
try {<br />
elem[ type ]();<br />
// prevent IE from throwing an error for some hidden elements<br />
} catch (e) {}<br />
}<br />
<br />
this.triggered = false;<br />
<br />
if ( !event.isPropagationStopped() ) {<br />
var parent = elem.parentNode || elem.ownerDocument;<br />
if ( parent ) {<br />
jQuery.event.trigger( event, data, parent, true );<br />
}<br />
}<br />
},<br />
<br />
handle: function( event ) {<br />
// returned undefined or false<br />
var all, handlers;<br />
<br />
event = arguments[0] = jQuery.event.fix( event || window.event );<br />
event.currentTarget = this;<br />
<br />
// Namespaced event handlers<br />
var namespaces = event.type.split(".");<br />
event.type = namespaces.shift();<br />
<br />
// Cache this now, all = true means, any handler<br />
all = !namespaces.length && !event.exclusive;<br />
<br />
var namespace = new RegExp("(^|\\.)" + namespaces.slice().sort().join(".*\\.") + "(\\.|$)");<br />
<br />
handlers = ( jQuery.data(this, "events") || {} )[ event.type ];<br />
<br />
for ( var j in handlers ) {<br />
var handler = handlers[ j ];<br />
<br />
// Filter the functions by class<br />
if ( all || namespace.test(handler.type) ) {<br />
// Pass in a reference to the handler function itself<br />
// So that we can later remove it<br />
event.handler = handler;<br />
event.data = handler.data;<br />
<br />
var ret = handler.apply( this, arguments );<br />
<br />
if ( ret !== undefined ) {<br />
event.result = ret;<br />
if ( ret === false ) {<br />
event.preventDefault();<br />
event.stopPropagation();<br />
}<br />
}<br />
<br />
if ( event.isImmediatePropagationStopped() ) {<br />
break;<br />
}<br />
<br />
}<br />
}<br />
},<br />
<br />
props: "altKey attrChange attrName bubbles button cancelable charCode clientX clientY ctrlKey currentTarget data detail eventPhase fromElement handler keyCode layerX layerY metaKey newValue offsetX offsetY originalTarget pageX pageY prevValue relatedNode relatedTarget screenX screenY shiftKey srcElement target toElement view wheelDelta which".split(" "),<br />
<br />
fix: function( event ) {<br />
if ( event[ expando ] ) {<br />
return event;<br />
}<br />
<br />
// store a copy of the original event object<br />
// and "clone" to set read-only properties<br />
var originalEvent = event;<br />
event = jQuery.Event( originalEvent );<br />
<br />
for ( var i = this.props.length, prop; i; ) {<br />
prop = this.props[ --i ];<br />
event[ prop ] = originalEvent[ prop ];<br />
}<br />
<br />
// Fix target property, if necessary<br />
if ( !event.target ) {<br />
event.target = event.srcElement || document; // Fixes #1925 where srcElement might not be defined either<br />
}<br />
<br />
// check if target is a textnode (safari)<br />
if ( event.target.nodeType === 3 ) {<br />
event.target = event.target.parentNode;<br />
}<br />
<br />
// Add relatedTarget, if necessary<br />
if ( !event.relatedTarget && event.fromElement ) {<br />
event.relatedTarget = event.fromElement === event.target ? event.toElement : event.fromElement;<br />
}<br />
<br />
// Calculate pageX/Y if missing and clientX/Y available<br />
if ( event.pageX == null && event.clientX != null ) {<br />
var doc = document.documentElement, body = document.body;<br />
event.pageX = event.clientX + (doc && doc.scrollLeft || body && body.scrollLeft || 0) - (doc && doc.clientLeft || body && body.clientLeft || 0);<br />
event.pageY = event.clientY + (doc && doc.scrollTop || body && body.scrollTop || 0) - (doc && doc.clientTop || body && body.clientTop || 0);<br />
}<br />
<br />
// Add which for key events<br />
if ( !event.which && ((event.charCode || event.charCode === 0) ? event.charCode : event.keyCode) ) {<br />
event.which = event.charCode || event.keyCode;<br />
}<br />
<br />
// Add metaKey to non-Mac browsers (use ctrl for PC's and Meta for Macs)<br />
if ( !event.metaKey && event.ctrlKey ) {<br />
event.metaKey = event.ctrlKey;<br />
}<br />
<br />
// Add which for click: 1 == left; 2 == middle; 3 == right<br />
// Note: button is not normalized, so don't use it<br />
if ( !event.which && event.button ) {<br />
event.which = (event.button & 1 ? 1 : ( event.button & 2 ? 3 : ( event.button & 4 ? 2 : 0 ) ));<br />
}<br />
<br />
return event;<br />
},<br />
<br />
proxy: function( fn, proxy, thisObject ) {<br />
if ( proxy !== undefined && !jQuery.isFunction( proxy ) ) {<br />
thisObject = proxy;<br />
proxy = undefined;<br />
}<br />
// FIXME: Should proxy be redefined to be applied with thisObject if defined?<br />
proxy = proxy || function() { return fn.apply( thisObject !== undefined ? thisObject : this, arguments ); };<br />
// Set the guid of unique handler to the same of original handler, so it can be removed<br />
proxy.guid = fn.guid = fn.guid || proxy.guid || this.guid++;<br />
// So proxy can be declared as an argument<br />
return proxy;<br />
},<br />
<br />
special: {<br />
ready: {<br />
// Make sure the ready event is setup<br />
setup: bindReady,<br />
teardown: function() {}<br />
},<br />
<br />
live: {<br />
add: function( proxy, data, namespaces ) {<br />
jQuery.extend( proxy, data || {} );<br />
proxy.guid += data.selector + data.live;<br />
jQuery.event.add( this, data.live, liveHandler );<br />
},<br />
<br />
remove: function( namespaces ) {<br />
if ( namespaces.length ) {<br />
var remove = 0, name = new RegExp("(^|\\.)" + namespaces[0] + "(\\.|$)");<br />
<br />
jQuery.each( (jQuery.data(this, "events").live || {}), function() {<br />
if ( name.test(this.type) ) {<br />
remove++;<br />
}<br />
});<br />
<br />
if ( remove < 1 ) {<br />
jQuery.event.remove( this, namespaces[0], liveHandler );<br />
}<br />
}<br />
}<br />
}<br />
}<br />
};<br />
<br />
jQuery.Event = function( src ){<br />
// Allow instantiation without the 'new' keyword<br />
if ( !this.preventDefault ) {<br />
return new jQuery.Event( src );<br />
}<br />
<br />
// Event object<br />
if ( src && src.type ) {<br />
this.originalEvent = src;<br />
this.type = src.type;<br />
// Event type<br />
} else {<br />
this.type = src;<br />
}<br />
<br />
// timeStamp is buggy for some events on Firefox(#3843)<br />
// So we won't rely on the native value<br />
this.timeStamp = now();<br />
<br />
// Mark it as fixed<br />
this[ expando ] = true;<br />
};<br />
<br />
function returnFalse() {<br />
return false;<br />
}<br />
function returnTrue() {<br />
return true;<br />
}<br />
<br />
// jQuery.Event is based on DOM3 Events as specified by the ECMAScript Language Binding<br />
// http://www.w3.org/TR/2003/WD-DOM-Level-3-Events-20030331/ecma-script-binding.html<br />
jQuery.Event.prototype = {<br />
preventDefault: function() {<br />
this.isDefaultPrevented = returnTrue;<br />
<br />
var e = this.originalEvent;<br />
if ( !e ) {<br />
return;<br />
}<br />
// if preventDefault exists run it on the original event<br />
if ( e.preventDefault ) {<br />
e.preventDefault();<br />
}<br />
// otherwise set the returnValue property of the original event to false (IE)<br />
e.returnValue = false;<br />
},<br />
stopPropagation: function() {<br />
this.isPropagationStopped = returnTrue;<br />
<br />
var e = this.originalEvent;<br />
if ( !e ) {<br />
return;<br />
}<br />
// if stopPropagation exists run it on the original event<br />
if ( e.stopPropagation ) {<br />
e.stopPropagation();<br />
}<br />
// otherwise set the cancelBubble property of the original event to true (IE)<br />
e.cancelBubble = true;<br />
},<br />
stopImmediatePropagation: function(){<br />
this.isImmediatePropagationStopped = returnTrue;<br />
this.stopPropagation();<br />
},<br />
isDefaultPrevented: returnFalse,<br />
isPropagationStopped: returnFalse,<br />
isImmediatePropagationStopped: returnFalse<br />
};<br />
// Checks if an event happened on an element within another element<br />
// Used in jQuery.event.special.mouseenter and mouseleave handlers<br />
var withinElement = function( event ) {<br />
// Check if mouse(over|out) are still within the same parent element<br />
var parent = event.relatedTarget;<br />
// Traverse up the tree<br />
while ( parent && parent != this ) {<br />
// Firefox sometimes assigns relatedTarget a XUL element<br />
// which we cannot access the parentNode property of<br />
try { parent = parent.parentNode; }<br />
// assuming we've left the element since we most likely mousedover a xul element<br />
catch(e) { break; }<br />
}<br />
<br />
if ( parent != this ) {<br />
// set the correct event type<br />
event.type = event.data;<br />
// handle event if we actually just moused on to a non sub-element<br />
jQuery.event.handle.apply( this, arguments );<br />
}<br />
};<br />
<br />
jQuery.each({<br />
mouseover: 'mouseenter',<br />
mouseout: 'mouseleave'<br />
}, function( orig, fix ) {<br />
jQuery.event.special[ fix ] = {<br />
setup: function(){<br />
jQuery.event.add( this, orig, withinElement, fix );<br />
},<br />
teardown: function(){<br />
jQuery.event.remove( this, orig, withinElement );<br />
}<br />
};<br />
});<br />
<br />
jQuery.fn.extend({<br />
bind: function( type, data, fn, thisObject ) {<br />
if ( jQuery.isFunction( data ) ) {<br />
if ( fn !== undefined ) {<br />
thisObject = fn;<br />
}<br />
fn = data;<br />
data = undefined;<br />
}<br />
fn = thisObject === undefined ? fn : jQuery.event.proxy( fn, thisObject );<br />
return type === "unload" ? this.one(type, data, fn, thisObject) : this.each(function() {<br />
jQuery.event.add( this, type, fn, data );<br />
});<br />
},<br />
<br />
one: function( type, data, fn, thisObject ) {<br />
if ( jQuery.isFunction( data ) ) {<br />
if ( fn !== undefined ) {<br />
thisObject = fn;<br />
}<br />
fn = data;<br />
data = undefined;<br />
}<br />
fn = thisObject === undefined ? fn : jQuery.event.proxy( fn, thisObject );<br />
var one = jQuery.event.proxy( fn, function( event ) {<br />
jQuery( this ).unbind( event, one );<br />
return fn.apply( this, arguments );<br />
});<br />
return this.each(function() {<br />
jQuery.event.add( this, type, one, data );<br />
});<br />
},<br />
<br />
unbind: function( type, fn ) {<br />
return this.each(function() {<br />
jQuery.event.remove( this, type, fn );<br />
});<br />
},<br />
<br />
trigger: function( type, data ) {<br />
return this.each(function() {<br />
jQuery.event.trigger( type, data, this );<br />
});<br />
},<br />
<br />
triggerHandler: function( type, data ) {<br />
if ( this[0] ) {<br />
var event = jQuery.Event( type );<br />
event.preventDefault();<br />
event.stopPropagation();<br />
jQuery.event.trigger( event, data, this[0] );<br />
return event.result;<br />
}<br />
},<br />
<br />
toggle: function( fn ) {<br />
// Save reference to arguments for access in closure<br />
var args = arguments, i = 1;<br />
<br />
// link all the functions, so any of them can unbind this click handler<br />
while( i < args.length ) {<br />
jQuery.event.proxy( fn, args[ i++ ] );<br />
}<br />
<br />
return this.click( jQuery.event.proxy( fn, function( event ) {<br />
// Figure out which function to execute<br />
this.lastToggle = ( this.lastToggle || 0 ) % i;<br />
<br />
// Make sure that clicks stop<br />
event.preventDefault();<br />
<br />
// and execute the function<br />
return args[ this.lastToggle++ ].apply( this, arguments ) || false;<br />
}));<br />
},<br />
<br />
hover: function( fnOver, fnOut ) {<br />
return this.mouseenter( fnOver ).mouseleave( fnOut || fnOver );<br />
},<br />
<br />
ready: function( fn ) {<br />
// Attach the listeners<br />
bindReady();<br />
<br />
// If the DOM is already ready<br />
if ( jQuery.isReady ) {<br />
// Execute the function immediately<br />
fn.call( document, jQuery );<br />
<br />
// Otherwise, remember the function for later<br />
} else {<br />
// Add the function to the wait list<br />
jQuery.readyList.push( fn );<br />
}<br />
<br />
return this;<br />
},<br />
<br />
live: function( type, data, fn, thisObject ) {<br />
if ( jQuery.isFunction( data ) ) {<br />
if ( fn !== undefined ) {<br />
thisObject = fn;<br />
}<br />
fn = data;<br />
data = undefined;<br />
}<br />
jQuery( this.context ).bind( liveConvert( type, this.selector ), {<br />
data: data, selector: this.selector, live: type<br />
}, fn, thisObject );<br />
return this;<br />
},<br />
<br />
die: function( type, fn ) {<br />
jQuery( this.context ).unbind( liveConvert( type, this.selector ), fn ? { guid: fn.guid + this.selector + type } : null );<br />
return this;<br />
}<br />
});<br />
<br />
function liveHandler( event ) {<br />
var stop = true, elems = [], args = arguments;<br />
<br />
jQuery.each( jQuery.data( this, "events" ).live || [], function( i, fn ) {<br />
if ( fn.live === event.type ) {<br />
var elem = jQuery( event.target ).closest( fn.selector )[0];<br />
if ( elem ) {<br />
elems.push({ elem: elem, fn: fn });<br />
}<br />
}<br />
});<br />
<br />
elems.sort(function( a, b ) {<br />
return jQuery.data( a.elem, "closest" ) - jQuery.data( b.elem, "closest" );<br />
});<br />
<br />
jQuery.each(elems, function() {<br />
event.currentTarget = this.elem;<br />
event.data = this.fn.data;<br />
if ( this.fn.apply( this.elem, args ) === false ) {<br />
return (stop = false);<br />
}<br />
});<br />
<br />
return stop;<br />
}<br />
<br />
function liveConvert( type, selector ) {<br />
return ["live", type, selector.replace(/\./g, "`").replace(/ /g, "|")].join(".");<br />
}<br />
<br />
jQuery.extend({<br />
isReady: false,<br />
readyList: [],<br />
// Handle when the DOM is ready<br />
ready: function() {<br />
// Make sure that the DOM is not already loaded<br />
if ( !jQuery.isReady ) {<br />
// Remember that the DOM is ready<br />
jQuery.isReady = true;<br />
<br />
// If there are functions bound, to execute<br />
if ( jQuery.readyList ) {<br />
// Execute all of them<br />
var fn, i = 0;<br />
while ( (fn = jQuery.readyList[ i++ ]) ) {<br />
fn.call( document, jQuery );<br />
}<br />
<br />
// Reset the list of functions<br />
jQuery.readyList = null;<br />
}<br />
<br />
// Trigger any bound ready events<br />
jQuery( document ).triggerHandler( "ready" );<br />
}<br />
}<br />
});<br />
<br />
var readyBound = false;<br />
<br />
function bindReady() {<br />
if ( readyBound ) return;<br />
readyBound = true;<br />
<br />
// Mozilla, Opera and webkit nightlies currently support this event<br />
if ( document.addEventListener ) {<br />
// Use the handy event callback<br />
document.addEventListener( "DOMContentLoaded", function() {<br />
document.removeEventListener( "DOMContentLoaded", arguments.callee, false );<br />
jQuery.ready();<br />
}, false );<br />
<br />
// If IE event model is used<br />
} else if ( document.attachEvent ) {<br />
// ensure firing before onload,<br />
// maybe late but safe also for iframes<br />
document.attachEvent("onreadystatechange", function() {<br />
if ( document.readyState === "complete" ) {<br />
document.detachEvent( "onreadystatechange", arguments.callee );<br />
jQuery.ready();<br />
}<br />
});<br />
<br />
// If IE and not an iframe<br />
// continually check to see if the document is ready<br />
if ( document.documentElement.doScroll && window === window.top ) (function() {<br />
if ( jQuery.isReady ) {<br />
return;<br />
}<br />
<br />
try {<br />
// If IE is used, use the trick by Diego Perini<br />
// http://javascript.nwbox.com/IEContentLoaded/<br />
document.documentElement.doScroll("left");<br />
} catch( error ) {<br />
setTimeout( arguments.callee, 0 );<br />
return;<br />
}<br />
<br />
// and execute any waiting functions<br />
jQuery.ready();<br />
})();<br />
}<br />
<br />
// A fallback to window.onload, that will always work<br />
jQuery.event.add( window, "load", jQuery.ready );<br />
}<br />
<br />
jQuery.each( ("blur,focus,load,resize,scroll,unload,click,dblclick," +<br />
"mousedown,mouseup,mousemove,mouseover,mouseout,mouseenter,mouseleave," +<br />
"change,select,submit,keydown,keypress,keyup,error").split(","), function( i, name ) {<br />
<br />
// Handle event binding<br />
jQuery.fn[ name ] = function( fn ) {<br />
return fn ? this.bind( name, fn ) : this.trigger( name );<br />
};<br />
});<br />
<br />
// Prevent memory leaks in IE<br />
// And prevent errors on refresh with events like mouseover in other browsers<br />
// Window isn't included so as not to unbind existing unload events<br />
// More info:<br />
// - http://isaacschlueter.com/2006/10/msie-memory-leaks/<br />
// - https://bugzilla.mozilla.org/show_bug.cgi?id=252542<br />
jQuery( window ).bind( 'unload', function() {<br />
for ( var id in jQuery.cache ) {<br />
// Skip the window<br />
if ( id != 1 && jQuery.cache[ id ].handle ) {<br />
jQuery.event.remove( jQuery.cache[ id ].handle.elem );<br />
}<br />
}<br />
});<br />
(function(){<br />
<br />
jQuery.support = {};<br />
<br />
var root = document.documentElement,<br />
script = document.createElement("script"),<br />
div = document.createElement("div"),<br />
id = "script" + (new Date).getTime();<br />
<br />
div.style.display = "none";<br />
div.innerHTML = ' <link/><table></table><a href="/a" style="color:red;float:left;opacity:.5;">a</a><select><option>text</option></select>';<br />
<br />
var all = div.getElementsByTagName("*"),<br />
a = div.getElementsByTagName("a")[0];<br />
<br />
// Can't get basic test support<br />
if ( !all || !all.length || !a ) {<br />
return;<br />
}<br />
<br />
jQuery.support = {<br />
// IE strips leading whitespace when .innerHTML is used<br />
leadingWhitespace: div.firstChild.nodeType == 3,<br />
<br />
// Make sure that tbody elements aren't automatically inserted<br />
// IE will insert them into empty tables<br />
tbody: !div.getElementsByTagName("tbody").length,<br />
<br />
// Make sure that link elements get serialized correctly by innerHTML<br />
// This requires a wrapper element in IE<br />
htmlSerialize: !!div.getElementsByTagName("link").length,<br />
<br />
// Get the style information from getAttribute<br />
// (IE uses .cssText insted)<br />
style: /red/.test( a.getAttribute("style") ),<br />
<br />
// Make sure that URLs aren't manipulated<br />
// (IE normalizes it by default)<br />
hrefNormalized: a.getAttribute("href") === "/a",<br />
<br />
// Make sure that element opacity exists<br />
// (IE uses filter instead)<br />
opacity: a.style.opacity === "0.5",<br />
<br />
// Verify style float existence<br />
// (IE uses styleFloat instead of cssFloat)<br />
cssFloat: !!a.style.cssFloat,<br />
<br />
// Will be defined later<br />
scriptEval: false,<br />
noCloneEvent: true,<br />
boxModel: null<br />
};<br />
<br />
script.type = "text/javascript";<br />
try {<br />
script.appendChild( document.createTextNode( "window." + id + "=1;" ) );<br />
} catch(e){}<br />
<br />
root.insertBefore( script, root.firstChild );<br />
<br />
// Make sure that the execution of code works by injecting a script<br />
// tag with appendChild/createTextNode<br />
// (IE doesn't support this, fails, and uses .text instead)<br />
if ( window[ id ] ) {<br />
jQuery.support.scriptEval = true;<br />
delete window[ id ];<br />
}<br />
<br />
root.removeChild( script );<br />
<br />
if ( div.attachEvent && div.fireEvent ) {<br />
div.attachEvent("onclick", function click(){<br />
// Cloning a node shouldn't copy over any<br />
// bound event handlers (IE does this)<br />
jQuery.support.noCloneEvent = false;<br />
div.detachEvent("onclick", click);<br />
});<br />
div.cloneNode(true).fireEvent("onclick");<br />
}<br />
<br />
// Figure out if the W3C box model works as expected<br />
// document.body must exist before we can do this<br />
jQuery(function(){<br />
var div = document.createElement("div");<br />
div.style.width = div.style.paddingLeft = "1px";<br />
<br />
document.body.appendChild( div );<br />
jQuery.boxModel = jQuery.support.boxModel = div.offsetWidth === 2;<br />
document.body.removeChild( div ).style.display = 'none';<br />
div = null;<br />
});<br />
<br />
// release memory in IE<br />
root = script = div = all = a = null;<br />
})();<br />
<br />
jQuery.props = {<br />
"for": "htmlFor",<br />
"class": "className",<br />
readonly: "readOnly",<br />
maxlength: "maxLength",<br />
cellspacing: "cellSpacing",<br />
rowspan: "rowSpan",<br />
colspan: "colSpan",<br />
tabindex: "tabIndex"<br />
};<br />
// exclude the following css properties to add px<br />
var exclude = /z-?index|font-?weight|opacity|zoom|line-?height/i,<br />
// cache check for defaultView.getComputedStyle<br />
getComputedStyle = document.defaultView && document.defaultView.getComputedStyle,<br />
// normalize float css property<br />
styleFloat = jQuery.support.cssFloat ? "cssFloat" : "styleFloat";<br />
<br />
jQuery.fn.css = function( name, value ) {<br />
var options = name, isFunction = jQuery.isFunction( value );<br />
<br />
if ( typeof name === "string" ) {<br />
// Are we setting the style?<br />
if ( value === undefined ) {<br />
return this.length ?<br />
jQuery.css( this[0], name ) :<br />
null;<br />
<br />
// Convert name, value params to options hash format<br />
} else {<br />
options = {};<br />
options[ name ] = value;<br />
}<br />
}<br />
<br />
// For each element...<br />
for ( var i = 0, l = this.length; i < l; i++ ) {<br />
var elem = this[i];<br />
<br />
// Set all the styles<br />
for ( var prop in options ) {<br />
value = options[prop];<br />
<br />
if ( isFunction ) {<br />
value = value.call( elem, i );<br />
}<br />
<br />
if ( typeof value === "number" && !exclude.test(prop) ) {<br />
value = value + "px";<br />
}<br />
<br />
jQuery.style( elem, prop, value );<br />
}<br />
}<br />
<br />
return this;<br />
};<br />
<br />
jQuery.extend({<br />
style: function( elem, name, value ) {<br />
// don't set styles on text and comment nodes<br />
if (!elem || elem.nodeType == 3 || elem.nodeType == 8)<br />
return undefined;<br />
<br />
// ignore negative width and height values #1599<br />
if ( (name == 'width' || name == 'height') && parseFloat(value) < 0 )<br />
value = undefined;<br />
<br />
var style = elem.style || elem, set = value !== undefined;<br />
<br />
// IE uses filters for opacity<br />
if ( !jQuery.support.opacity && name == "opacity" ) {<br />
if ( set ) {<br />
// IE has trouble with opacity if it does not have layout<br />
// Force it by setting the zoom level<br />
style.zoom = 1;<br />
<br />
// Set the alpha filter to set the opacity<br />
style.filter = (style.filter || "").replace( /alpha\([^)]*\)/, "" ) +<br />
(parseInt( value ) + '' == "NaN" ? "" : "alpha(opacity=" + value * 100 + ")");<br />
}<br />
<br />
return style.filter && style.filter.indexOf("opacity=") >= 0 ?<br />
(parseFloat( style.filter.match(/opacity=([^)]*)/)[1] ) / 100) + '':<br />
"";<br />
}<br />
<br />
// Make sure we're using the right name for getting the float value<br />
if ( /float/i.test( name ) )<br />
name = styleFloat;<br />
<br />
name = name.replace(/-([a-z])/ig, function(all, letter){<br />
return letter.toUpperCase();<br />
});<br />
<br />
if ( set )<br />
style[ name ] = value;<br />
<br />
return style[ name ];<br />
},<br />
<br />
css: function( elem, name, force, extra ) {<br />
if ( name == "width" || name == "height" ) {<br />
var val, props = { position: "absolute", visibility: "hidden", display:"block" }, which = name == "width" ? [ "Left", "Right" ] : [ "Top", "Bottom" ];<br />
<br />
function getWH() {<br />
val = name == "width" ? elem.offsetWidth : elem.offsetHeight;<br />
<br />
if ( extra === "border" )<br />
return;<br />
<br />
jQuery.each( which, function() {<br />
if ( !extra )<br />
val -= parseFloat(jQuery.curCSS( elem, "padding" + this, true)) || 0;<br />
if ( extra === "margin" )<br />
val += parseFloat(jQuery.curCSS( elem, "margin" + this, true)) || 0;<br />
else<br />
val -= parseFloat(jQuery.curCSS( elem, "border" + this + "Width", true)) || 0;<br />
});<br />
}<br />
<br />
if ( elem.offsetWidth !== 0 )<br />
getWH();<br />
else<br />
jQuery.swap( elem, props, getWH );<br />
<br />
return Math.max(0, Math.round(val));<br />
}<br />
<br />
return jQuery.curCSS( elem, name, force );<br />
},<br />
<br />
curCSS: function( elem, name, force ) {<br />
var ret, style = elem.style, filter;<br />
<br />
// IE uses filters for opacity<br />
if ( !jQuery.support.opacity && name === "opacity" && elem.currentStyle ) {<br />
ret = (elem.currentStyle.filter || "").match(/opacity=([^)]*)/) ?<br />
(parseFloat(RegExp.$1) / 100) + "" :<br />
"";<br />
<br />
return ret === "" ?<br />
"1" :<br />
ret;<br />
}<br />
<br />
// Make sure we're using the right name for getting the float value<br />
if ( /float/i.test( name ) )<br />
name = styleFloat;<br />
<br />
if ( !force && style && style[ name ] ) {<br />
ret = style[ name ];<br />
<br />
} else if ( getComputedStyle ) {<br />
<br />
// Only "float" is needed here<br />
if ( /float/i.test( name ) )<br />
name = "float";<br />
<br />
name = name.replace( /([A-Z])/g, "-$1" ).toLowerCase();<br />
<br />
var computedStyle = elem.ownerDocument.defaultView.getComputedStyle( elem, null );<br />
<br />
if ( computedStyle )<br />
ret = computedStyle.getPropertyValue( name );<br />
<br />
// We should always get a number back from opacity<br />
if ( name == "opacity" && ret == "" )<br />
ret = "1";<br />
<br />
} else if ( elem.currentStyle ) {<br />
var camelCase = name.replace(/\-(\w)/g, function(all, letter){<br />
return letter.toUpperCase();<br />
});<br />
<br />
ret = elem.currentStyle[ name ] || elem.currentStyle[ camelCase ];<br />
<br />
// From the awesome hack by Dean Edwards<br />
// http://erik.eae.net/archives/2007/07/27/18.54.15/#comment-102291<br />
<br />
// If we're not dealing with a regular pixel number<br />
// but a number that has a weird ending, we need to convert it to pixels<br />
if ( !/^\d+(px)?$/i.test( ret ) && /^\d/.test( ret ) ) {<br />
// Remember the original values<br />
var left = style.left, rsLeft = elem.runtimeStyle.left;<br />
<br />
// Put in the new values to get a computed value out<br />
elem.runtimeStyle.left = elem.currentStyle.left;<br />
style.left = ret || 0;<br />
ret = style.pixelLeft + "px";<br />
<br />
// Revert the changed values<br />
style.left = left;<br />
elem.runtimeStyle.left = rsLeft;<br />
}<br />
}<br />
<br />
return ret;<br />
},<br />
<br />
// A method for quickly swapping in/out CSS properties to get correct calculations<br />
swap: function( elem, options, callback ) {<br />
var old = {};<br />
// Remember the old values, and insert the new ones<br />
for ( var name in options ) {<br />
old[ name ] = elem.style[ name ];<br />
elem.style[ name ] = options[ name ];<br />
}<br />
<br />
callback.call( elem );<br />
<br />
// Revert the old values<br />
for ( var name in options )<br />
elem.style[ name ] = old[ name ];<br />
}<br />
});jQuery.fn.extend({<br />
// Keep a copy of the old load<br />
_load: jQuery.fn.load,<br />
<br />
load: function( url, params, callback ) {<br />
if ( typeof url !== "string" )<br />
return this._load( url );<br />
<br />
var off = url.indexOf(" ");<br />
if ( off >= 0 ) {<br />
var selector = url.slice(off, url.length);<br />
url = url.slice(0, off);<br />
}<br />
<br />
// Default to a GET request<br />
var type = "GET";<br />
<br />
// If the second parameter was provided<br />
if ( params )<br />
// If it's a function<br />
if ( jQuery.isFunction( params ) ) {<br />
// We assume that it's the callback<br />
callback = params;<br />
params = null;<br />
<br />
// Otherwise, build a param string<br />
} else if( typeof params === "object" ) {<br />
params = jQuery.param( params );<br />
type = "POST";<br />
}<br />
<br />
var self = this;<br />
<br />
// Request the remote document<br />
jQuery.ajax({<br />
url: url,<br />
type: type,<br />
dataType: "html",<br />
data: params,<br />
complete: function(res, status){<br />
// If successful, inject the HTML into all the matched elements<br />
if ( status == "success" || status == "notmodified" )<br />
// See if a selector was specified<br />
self.html( selector ?<br />
// Create a dummy div to hold the results<br />
jQuery("<div/>")<br />
// inject the contents of the document in, removing the scripts<br />
// to avoid any 'Permission Denied' errors in IE<br />
.append(res.responseText.replace(/<script(.|\s)*?\/script>/g, ""))<br />
<br />
// Locate the specified elements<br />
.find(selector) :<br />
<br />
// If not, just inject the full result<br />
res.responseText );<br />
<br />
if( callback )<br />
self.each( callback, [res.responseText, status, res] );<br />
}<br />
});<br />
return this;<br />
},<br />
<br />
serialize: function() {<br />
return jQuery.param(this.serializeArray());<br />
},<br />
serializeArray: function() {<br />
return this.map(function(){<br />
return this.elements ? jQuery.makeArray(this.elements) : this;<br />
})<br />
.filter(function(){<br />
return this.name && !this.disabled &&<br />
(this.checked || /select|textarea/i.test(this.nodeName) ||<br />
/text|hidden|password|search/i.test(this.type));<br />
})<br />
.map(function(i, elem){<br />
var val = jQuery(this).val();<br />
return val == null ? null :<br />
jQuery.isArray(val) ?<br />
jQuery.map( val, function(val, i){<br />
return {name: elem.name, value: val};<br />
}) :<br />
{name: elem.name, value: val};<br />
}).get();<br />
}<br />
});<br />
<br />
// Attach a bunch of functions for handling common AJAX events<br />
jQuery.each( "ajaxStart,ajaxStop,ajaxComplete,ajaxError,ajaxSuccess,ajaxSend".split(","), function(i,o){<br />
jQuery.fn[o] = function(f){<br />
return this.bind(o, f);<br />
};<br />
});<br />
<br />
var jsc = now();<br />
<br />
jQuery.extend({<br />
<br />
get: function( url, data, callback, type ) {<br />
// shift arguments if data argument was ommited<br />
if ( jQuery.isFunction( data ) ) {<br />
callback = data;<br />
data = null;<br />
}<br />
<br />
return jQuery.ajax({<br />
type: "GET",<br />
url: url,<br />
data: data,<br />
success: callback,<br />
dataType: type<br />
});<br />
},<br />
<br />
getScript: function( url, callback ) {<br />
return jQuery.get(url, null, callback, "script");<br />
},<br />
<br />
getJSON: function( url, data, callback ) {<br />
return jQuery.get(url, data, callback, "json");<br />
},<br />
<br />
post: function( url, data, callback, type ) {<br />
if ( jQuery.isFunction( data ) ) {<br />
callback = data;<br />
data = {};<br />
}<br />
<br />
return jQuery.ajax({<br />
type: "POST",<br />
url: url,<br />
data: data,<br />
success: callback,<br />
dataType: type<br />
});<br />
},<br />
<br />
ajaxSetup: function( settings ) {<br />
jQuery.extend( jQuery.ajaxSettings, settings );<br />
},<br />
<br />
ajaxSettings: {<br />
url: location.href,<br />
global: true,<br />
type: "GET",<br />
contentType: "application/x-www-form-urlencoded",<br />
processData: true,<br />
async: true,<br />
/*<br />
timeout: 0,<br />
data: null,<br />
username: null,<br />
password: null,<br />
*/<br />
// Create the request object; Microsoft failed to properly<br />
// implement the XMLHttpRequest in IE7, so we use the ActiveXObject when it is available<br />
// This function can be overriden by calling jQuery.ajaxSetup<br />
xhr:function(){<br />
return window.ActiveXObject ? new ActiveXObject("Microsoft.XMLHTTP") : new XMLHttpRequest();<br />
},<br />
accepts: {<br />
xml: "application/xml, text/xml",<br />
html: "text/html",<br />
script: "text/javascript, application/javascript",<br />
json: "application/json, text/javascript",<br />
text: "text/plain",<br />
_default: "*/*"<br />
}<br />
},<br />
<br />
// Last-Modified header cache for next request<br />
lastModified: {},<br />
<br />
ajax: function( s ) {<br />
// Extend the settings, but re-extend 's' so that it can be<br />
// checked again later (in the test suite, specifically)<br />
s = jQuery.extend(true, s, jQuery.extend(true, {}, jQuery.ajaxSettings, s));<br />
<br />
var jsonp, jsre = /=\?(&|$)/g, status, data,<br />
type = s.type.toUpperCase();<br />
<br />
// convert data if not already a string<br />
if ( s.data && s.processData && typeof s.data !== "string" )<br />
s.data = jQuery.param(s.data);<br />
<br />
// Handle JSONP Parameter Callbacks<br />
if ( s.dataType == "jsonp" ) {<br />
if ( type == "GET" ) {<br />
if ( !s.url.match(jsre) )<br />
s.url += (s.url.match(/\?/) ? "&" : "?") + (s.jsonp || "callback") + "=?";<br />
} else if ( !s.data || !s.data.match(jsre) )<br />
s.data = (s.data ? s.data + "&" : "") + (s.jsonp || "callback") + "=?";<br />
s.dataType = "json";<br />
}<br />
<br />
// Build temporary JSONP function<br />
if ( s.dataType == "json" && (s.data && s.data.match(jsre) || s.url.match(jsre)) ) {<br />
jsonp = "jsonp" + jsc++;<br />
<br />
// Replace the =? sequence both in the query string and the data<br />
if ( s.data )<br />
s.data = (s.data + "").replace(jsre, "=" + jsonp + "$1");<br />
s.url = s.url.replace(jsre, "=" + jsonp + "$1");<br />
<br />
// We need to make sure<br />
// that a JSONP style response is executed properly<br />
s.dataType = "script";<br />
<br />
// Handle JSONP-style loading<br />
window[ jsonp ] = function(tmp){<br />
data = tmp;<br />
success();<br />
complete();<br />
// Garbage collect<br />
window[ jsonp ] = undefined;<br />
try{ delete window[ jsonp ]; } catch(e){}<br />
if ( head )<br />
head.removeChild( script );<br />
};<br />
}<br />
<br />
if ( s.dataType == "script" && s.cache == null )<br />
s.cache = false;<br />
<br />
if ( s.cache === false && type == "GET" ) {<br />
var ts = now();<br />
// try replacing _= if it is there<br />
var ret = s.url.replace(/(\?|&)_=.*?(&|$)/, "$1_=" + ts + "$2");<br />
// if nothing was replaced, add timestamp to the end<br />
s.url = ret + ((ret == s.url) ? (s.url.match(/\?/) ? "&" : "?") + "_=" + ts : "");<br />
}<br />
<br />
// If data is available, append data to url for get requests<br />
if ( s.data && type == "GET" ) {<br />
s.url += (s.url.match(/\?/) ? "&" : "?") + s.data;<br />
}<br />
<br />
// Watch for a new set of requests<br />
if ( s.global && ! jQuery.active++ )<br />
jQuery.event.trigger( "ajaxStart" );<br />
<br />
// Matches an absolute URL, and saves the domain<br />
var parts = /^(\w+:)?\/\/([^\/?#]+)/.exec( s.url );<br />
<br />
// If we're requesting a remote document<br />
// and trying to load JSON or Script with a GET<br />
if ( s.dataType == "script" && type == "GET" && parts<br />
&& ( parts[1] && parts[1] != location.protocol || parts[2] != location.host )){<br />
<br />
var head = document.getElementsByTagName("head")[0];<br />
var script = document.createElement("script");<br />
script.src = s.url;<br />
if (s.scriptCharset)<br />
script.charset = s.scriptCharset;<br />
<br />
// Handle Script loading<br />
if ( !jsonp ) {<br />
var done = false;<br />
<br />
// Attach handlers for all browsers<br />
script.onload = script.onreadystatechange = function(){<br />
if ( !done && (!this.readyState ||<br />
this.readyState == "loaded" || this.readyState == "complete") ) {<br />
done = true;<br />
success();<br />
complete();<br />
<br />
// Handle memory leak in IE<br />
script.onload = script.onreadystatechange = null;<br />
head.removeChild( script );<br />
}<br />
};<br />
}<br />
<br />
// Use insertBefore instead of appendChild to circumvent an IE6 bug.<br />
// This arises when a base node is used (#2709 and #4378).<br />
head.insertBefore( script, head.firstChild );<br />
<br />
// We handle everything using the script element injection<br />
return undefined;<br />
}<br />
<br />
var requestDone = false;<br />
<br />
// Create the request object<br />
var xhr = s.xhr();<br />
<br />
// Open the socket<br />
// Passing null username, generates a login popup on Opera (#2865)<br />
if( s.username )<br />
xhr.open(type, s.url, s.async, s.username, s.password);<br />
else<br />
xhr.open(type, s.url, s.async);<br />
<br />
// Need an extra try/catch for cross domain requests in Firefox 3<br />
try {<br />
// Set the correct header, if data is being sent<br />
if ( s.data )<br />
xhr.setRequestHeader("Content-Type", s.contentType);<br />
<br />
// Set the If-Modified-Since header, if ifModified mode.<br />
if ( s.ifModified )<br />
xhr.setRequestHeader("If-Modified-Since",<br />
jQuery.lastModified[s.url] || "Thu, 01 Jan 1970 00:00:00 GMT" );<br />
<br />
// Set header so the called script knows that it's an XMLHttpRequest<br />
xhr.setRequestHeader("X-Requested-With", "XMLHttpRequest");<br />
<br />
// Set the Accepts header for the server, depending on the dataType<br />
xhr.setRequestHeader("Accept", s.dataType && s.accepts[ s.dataType ] ?<br />
s.accepts[ s.dataType ] + ", */*" :<br />
s.accepts._default );<br />
} catch(e){}<br />
<br />
// Allow custom headers/mimetypes and early abort<br />
if ( s.beforeSend && s.beforeSend(xhr, s) === false ) {<br />
// Handle the global AJAX counter<br />
if ( s.global && ! --jQuery.active )<br />
jQuery.event.trigger( "ajaxStop" );<br />
// close opended socket<br />
xhr.abort();<br />
return false;<br />
}<br />
<br />
if ( s.global )<br />
jQuery.event.trigger("ajaxSend", [xhr, s]);<br />
<br />
// Wait for a response to come back<br />
var onreadystatechange = function(isTimeout){<br />
// The request was aborted, clear the interval and decrement jQuery.active<br />
if (xhr.readyState == 0) {<br />
if (ival) {<br />
// clear poll interval<br />
clearInterval(ival);<br />
ival = null;<br />
// Handle the global AJAX counter<br />
if ( s.global && ! --jQuery.active )<br />
jQuery.event.trigger( "ajaxStop" );<br />
}<br />
// The transfer is complete and the data is available, or the request timed out<br />
} else if ( !requestDone && xhr && (xhr.readyState == 4 || isTimeout == "timeout") ) {<br />
requestDone = true;<br />
<br />
// clear poll interval<br />
if (ival) {<br />
clearInterval(ival);<br />
ival = null;<br />
}<br />
<br />
status = isTimeout == "timeout" ? "timeout" :<br />
!jQuery.httpSuccess( xhr ) ? "error" :<br />
s.ifModified && jQuery.httpNotModified( xhr, s.url ) ? "notmodified" :<br />
"success";<br />
<br />
if ( status == "success" ) {<br />
// Watch for, and catch, XML document parse errors<br />
try {<br />
// process the data (runs the xml through httpData regardless of callback)<br />
data = jQuery.httpData( xhr, s.dataType, s );<br />
} catch(e) {<br />
status = "parsererror";<br />
}<br />
}<br />
<br />
// Make sure that the request was successful or notmodified<br />
if ( status == "success" ) {<br />
// Cache Last-Modified header, if ifModified mode.<br />
var modRes;<br />
try {<br />
modRes = xhr.getResponseHeader("Last-Modified");<br />
} catch(e) {} // swallow exception thrown by FF if header is not available<br />
<br />
if ( s.ifModified && modRes )<br />
jQuery.lastModified[s.url] = modRes;<br />
<br />
// JSONP handles its own success callback<br />
if ( !jsonp )<br />
success();<br />
} else<br />
jQuery.handleError(s, xhr, status);<br />
<br />
// Fire the complete handlers<br />
complete();<br />
<br />
if ( isTimeout )<br />
xhr.abort();<br />
<br />
// Stop memory leaks<br />
if ( s.async )<br />
xhr = null;<br />
}<br />
};<br />
<br />
if ( s.async ) {<br />
// don't attach the handler to the request, just poll it instead<br />
var ival = setInterval(onreadystatechange, 13);<br />
<br />
// Timeout checker<br />
if ( s.timeout > 0 )<br />
setTimeout(function(){<br />
// Check to see if the request is still happening<br />
if ( xhr && !requestDone )<br />
onreadystatechange( "timeout" );<br />
}, s.timeout);<br />
}<br />
<br />
// Send the data<br />
try {<br />
xhr.send( type === "POST" ? s.data : null );<br />
} catch(e) {<br />
jQuery.handleError(s, xhr, null, e);<br />
}<br />
<br />
// firefox 1.5 doesn't fire statechange for sync requests<br />
if ( !s.async )<br />
onreadystatechange();<br />
<br />
function success(){<br />
// If a local callback was specified, fire it and pass it the data<br />
if ( s.success )<br />
s.success( data, status );<br />
<br />
// Fire the global callback<br />
if ( s.global )<br />
jQuery.event.trigger( "ajaxSuccess", [xhr, s] );<br />
}<br />
<br />
function complete(){<br />
// Process result<br />
if ( s.complete )<br />
s.complete(xhr, status);<br />
<br />
// The request was completed<br />
if ( s.global )<br />
jQuery.event.trigger( "ajaxComplete", [xhr, s] );<br />
<br />
// Handle the global AJAX counter<br />
if ( s.global && ! --jQuery.active )<br />
jQuery.event.trigger( "ajaxStop" );<br />
}<br />
<br />
// return XMLHttpRequest to allow aborting the request etc.<br />
return xhr;<br />
},<br />
<br />
handleError: function( s, xhr, status, e ) {<br />
// If a local callback was specified, fire it<br />
if ( s.error ) s.error( xhr, status, e );<br />
<br />
// Fire the global callback<br />
if ( s.global )<br />
jQuery.event.trigger( "ajaxError", [xhr, s, e] );<br />
},<br />
<br />
// Counter for holding the number of active queries<br />
active: 0,<br />
<br />
// Determines if an XMLHttpRequest was successful or not<br />
httpSuccess: function( xhr ) {<br />
try {<br />
// IE error sometimes returns 1223 when it should be 204 so treat it as success, see #1450<br />
return !xhr.status && location.protocol == "file:" ||<br />
( xhr.status >= 200 && xhr.status < 300 ) || xhr.status == 304 || xhr.status == 1223;<br />
} catch(e){}<br />
return false;<br />
},<br />
<br />
// Determines if an XMLHttpRequest returns NotModified<br />
httpNotModified: function( xhr, url ) {<br />
try {<br />
var xhrRes = xhr.getResponseHeader("Last-Modified");<br />
<br />
// Firefox always returns 200. check Last-Modified date<br />
return xhr.status == 304 || xhrRes == jQuery.lastModified[url];<br />
} catch(e){}<br />
return false;<br />
},<br />
<br />
httpData: function( xhr, type, s ) {<br />
var ct = xhr.getResponseHeader("content-type"),<br />
xml = type == "xml" || !type && ct && ct.indexOf("xml") >= 0,<br />
data = xml ? xhr.responseXML : xhr.responseText;<br />
<br />
if ( xml && data.documentElement.tagName == "parsererror" ) {<br />
throw "parsererror";<br />
}<br />
<br />
// Allow a pre-filtering function to sanitize the response<br />
// s != null is checked to keep backwards compatibility<br />
if ( s && s.dataFilter ) {<br />
data = s.dataFilter( data, type );<br />
}<br />
<br />
// The filter can actually parse the response<br />
if ( typeof data === "string" ) {<br />
<br />
// If the type is "script", eval it in global context<br />
if ( type === "script" ) {<br />
jQuery.globalEval( data );<br />
}<br />
<br />
// Get the JavaScript object, if JSON is used.<br />
if ( type == "json" ) {<br />
if ( typeof JSON === "object" && JSON.parse ) {<br />
data = JSON.parse( data );<br />
} else {<br />
data = (new Function("return " + data))();<br />
}<br />
}<br />
}<br />
<br />
return data;<br />
},<br />
<br />
// Serialize an array of form elements or a set of<br />
// key/values into a query string<br />
param: function( a ) {<br />
var s = [ ];<br />
<br />
function add( key, value ){<br />
s[ s.length ] = encodeURIComponent(key) + '=' + encodeURIComponent(value);<br />
};<br />
<br />
// If an array was passed in, assume that it is an array<br />
// of form elements<br />
if ( jQuery.isArray(a) || a.jquery )<br />
// Serialize the form elements<br />
jQuery.each( a, function(){<br />
add( this.name, this.value );<br />
});<br />
<br />
// Otherwise, assume that it's an object of key/value pairs<br />
else<br />
// Serialize the key/values<br />
for ( var j in a )<br />
// If the value is an array then the key names need to be repeated<br />
if ( jQuery.isArray(a[j]) )<br />
jQuery.each( a[j], function(){<br />
add( j, this );<br />
});<br />
else<br />
add( j, jQuery.isFunction(a[j]) ? a[j]() : a[j] );<br />
<br />
// Return the resulting serialization<br />
return s.join("&").replace(/%20/g, "+");<br />
}<br />
<br />
});<br />
var elemdisplay = {},<br />
timerId,<br />
fxAttrs = [<br />
// height animations<br />
[ "height", "marginTop", "marginBottom", "paddingTop", "paddingBottom" ],<br />
// width animations<br />
[ "width", "marginLeft", "marginRight", "paddingLeft", "paddingRight" ],<br />
// opacity animations<br />
[ "opacity" ]<br />
];<br />
<br />
function genFx( type, num ){<br />
var obj = {};<br />
jQuery.each( fxAttrs.concat.apply([], fxAttrs.slice(0,num)), function(){<br />
obj[ this ] = type;<br />
});<br />
return obj;<br />
}<br />
<br />
jQuery.fn.extend({<br />
show: function(speed,callback){<br />
if ( speed ) {<br />
return this.animate( genFx("show", 3), speed, callback);<br />
} else {<br />
for ( var i = 0, l = this.length; i < l; i++ ){<br />
var old = jQuery.data(this[i], "olddisplay");<br />
<br />
this[i].style.display = old || "";<br />
<br />
if ( jQuery.css(this[i], "display") === "none" ) {<br />
var tagName = this[i].tagName, display;<br />
<br />
if ( elemdisplay[ tagName ] ) {<br />
display = elemdisplay[ tagName ];<br />
} else {<br />
var elem = jQuery("<" + tagName + " />").appendTo("body");<br />
<br />
display = elem.css("display");<br />
if ( display === "none" )<br />
display = "block";<br />
<br />
elem.remove();<br />
<br />
elemdisplay[ tagName ] = display;<br />
}<br />
<br />
jQuery.data(this[i], "olddisplay", display);<br />
}<br />
}<br />
<br />
// Set the display of the elements in a second loop<br />
// to avoid the constant reflow<br />
for ( var i = 0, l = this.length; i < l; i++ ){<br />
this[i].style.display = jQuery.data(this[i], "olddisplay") || "";<br />
}<br />
<br />
return this;<br />
}<br />
},<br />
<br />
hide: function(speed,callback){<br />
if ( speed ) {<br />
return this.animate( genFx("hide", 3), speed, callback);<br />
} else {<br />
for ( var i = 0, l = this.length; i < l; i++ ){<br />
var old = jQuery.data(this[i], "olddisplay");<br />
if ( !old && old !== "none" )<br />
jQuery.data(this[i], "olddisplay", jQuery.css(this[i], "display"));<br />
}<br />
<br />
// Set the display of the elements in a second loop<br />
// to avoid the constant reflow<br />
for ( var i = 0, l = this.length; i < l; i++ ){<br />
this[i].style.display = "none";<br />
}<br />
<br />
return this;<br />
}<br />
},<br />
<br />
// Save the old toggle function<br />
_toggle: jQuery.fn.toggle,<br />
<br />
toggle: function( fn, fn2 ){<br />
var bool = typeof fn === "boolean";<br />
<br />
return jQuery.isFunction(fn) && jQuery.isFunction(fn2) ?<br />
this._toggle.apply( this, arguments ) :<br />
fn == null || bool ?<br />
this.each(function(){<br />
var state = bool ? fn : jQuery(this).is(":hidden");<br />
jQuery(this)[ state ? "show" : "hide" ]();<br />
}) :<br />
this.animate(genFx("toggle", 3), fn, fn2);<br />
},<br />
<br />
fadeTo: function(speed,to,callback){<br />
return this.filter(":hidden").css('opacity', 0).show().end()<br />
.animate({opacity: to}, speed, callback);<br />
},<br />
<br />
animate: function( prop, speed, easing, callback ) {<br />
var optall = jQuery.speed(speed, easing, callback);<br />
<br />
return this[ optall.queue === false ? "each" : "queue" ](function(){<br />
<br />
var opt = jQuery.extend({}, optall), p,<br />
hidden = this.nodeType == 1 && jQuery(this).is(":hidden"),<br />
self = this;<br />
<br />
for ( p in prop ) {<br />
if ( prop[p] == "hide" && hidden || prop[p] == "show" && !hidden )<br />
return opt.complete.call(this);<br />
<br />
if ( ( p == "height" || p == "width" ) && this.style ) {<br />
// Store display property<br />
opt.display = jQuery.css(this, "display");<br />
<br />
// Make sure that nothing sneaks out<br />
opt.overflow = this.style.overflow;<br />
}<br />
}<br />
<br />
if ( opt.overflow != null )<br />
this.style.overflow = "hidden";<br />
<br />
opt.curAnim = jQuery.extend({}, prop);<br />
<br />
jQuery.each( prop, function(name, val){<br />
var e = new jQuery.fx( self, opt, name );<br />
<br />
if ( /toggle|show|hide/.test(val) )<br />
e[ val == "toggle" ? hidden ? "show" : "hide" : val ]( prop );<br />
else {<br />
var parts = val.toString().match(/^([+-]=)?([\d+-.]+)(.*)$/),<br />
start = e.cur(true) || 0;<br />
<br />
if ( parts ) {<br />
var end = parseFloat(parts[2]),<br />
unit = parts[3] || "px";<br />
<br />
// We need to compute starting value<br />
if ( unit != "px" ) {<br />
self.style[ name ] = (end || 1) + unit;<br />
start = ((end || 1) / e.cur(true)) * start;<br />
self.style[ name ] = start + unit;<br />
}<br />
<br />
// If a +=/-= token was provided, we're doing a relative animation<br />
if ( parts[1] )<br />
end = ((parts[1] == "-=" ? -1 : 1) * end) + start;<br />
<br />
e.custom( start, end, unit );<br />
} else<br />
e.custom( start, val, "" );<br />
}<br />
});<br />
<br />
// For JS strict compliance<br />
return true;<br />
});<br />
},<br />
<br />
stop: function(clearQueue, gotoEnd){<br />
var timers = jQuery.timers;<br />
<br />
if (clearQueue)<br />
this.queue([]);<br />
<br />
this.each(function(){<br />
// go in reverse order so anything added to the queue during the loop is ignored<br />
for ( var i = timers.length - 1; i >= 0; i-- )<br />
if ( timers[i].elem == this ) {<br />
if (gotoEnd)<br />
// force the next step to be the last<br />
timers[i](true);<br />
timers.splice(i, 1);<br />
}<br />
});<br />
<br />
// start the next in the queue if the last step wasn't forced<br />
if (!gotoEnd)<br />
this.dequeue();<br />
<br />
return this;<br />
}<br />
<br />
});<br />
<br />
// Generate shortcuts for custom animations<br />
jQuery.each({<br />
slideDown: genFx("show", 1),<br />
slideUp: genFx("hide", 1),<br />
slideToggle: genFx("toggle", 1),<br />
fadeIn: { opacity: "show" },<br />
fadeOut: { opacity: "hide" }<br />
}, function( name, props ){<br />
jQuery.fn[ name ] = function( speed, callback ){<br />
return this.animate( props, speed, callback );<br />
};<br />
});<br />
<br />
jQuery.extend({<br />
<br />
speed: function(speed, easing, fn) {<br />
var opt = typeof speed === "object" ? speed : {<br />
complete: fn || !fn && easing ||<br />
jQuery.isFunction( speed ) && speed,<br />
duration: speed,<br />
easing: fn && easing || easing && !jQuery.isFunction(easing) && easing<br />
};<br />
<br />
opt.duration = jQuery.fx.off ? 0 : typeof opt.duration === "number" ? opt.duration :<br />
jQuery.fx.speeds[opt.duration] || jQuery.fx.speeds._default;<br />
<br />
// Queueing<br />
opt.old = opt.complete;<br />
opt.complete = function(){<br />
if ( opt.queue !== false )<br />
jQuery(this).dequeue();<br />
if ( jQuery.isFunction( opt.old ) )<br />
opt.old.call( this );<br />
};<br />
<br />
return opt;<br />
},<br />
<br />
easing: {<br />
linear: function( p, n, firstNum, diff ) {<br />
return firstNum + diff * p;<br />
},<br />
swing: function( p, n, firstNum, diff ) {<br />
return ((-Math.cos(p*Math.PI)/2) + 0.5) * diff + firstNum;<br />
}<br />
},<br />
<br />
timers: [],<br />
<br />
fx: function( elem, options, prop ){<br />
this.options = options;<br />
this.elem = elem;<br />
this.prop = prop;<br />
<br />
if ( !options.orig )<br />
options.orig = {};<br />
}<br />
<br />
});<br />
<br />
jQuery.fx.prototype = {<br />
<br />
// Simple function for setting a style value<br />
update: function(){<br />
if ( this.options.step )<br />
this.options.step.call( this.elem, this.now, this );<br />
<br />
(jQuery.fx.step[this.prop] || jQuery.fx.step._default)( this );<br />
<br />
// Set display property to block for height/width animations<br />
if ( ( this.prop == "height" || this.prop == "width" ) && this.elem.style )<br />
this.elem.style.display = "block";<br />
},<br />
<br />
// Get the current size<br />
cur: function(force){<br />
if ( this.elem[this.prop] != null && (!this.elem.style || this.elem.style[this.prop] == null) )<br />
return this.elem[ this.prop ];<br />
<br />
var r = parseFloat(jQuery.css(this.elem, this.prop, force));<br />
return r && r > -10000 ? r : parseFloat(jQuery.curCSS(this.elem, this.prop)) || 0;<br />
},<br />
<br />
// Start an animation from one number to another<br />
custom: function(from, to, unit){<br />
this.startTime = now();<br />
this.start = from;<br />
this.end = to;<br />
this.unit = unit || this.unit || "px";<br />
this.now = this.start;<br />
this.pos = this.state = 0;<br />
<br />
var self = this;<br />
function t(gotoEnd){<br />
return self.step(gotoEnd);<br />
}<br />
<br />
t.elem = this.elem;<br />
<br />
if ( t() && jQuery.timers.push(t) && !timerId )<br />
timerId = setInterval(jQuery.fx.tick, 13);<br />
},<br />
<br />
// Simple 'show' function<br />
show: function(){<br />
// Remember where we started, so that we can go back to it later<br />
this.options.orig[this.prop] = jQuery.style( this.elem, this.prop );<br />
this.options.show = true;<br />
<br />
// Begin the animation<br />
// Make sure that we start at a small width/height to avoid any<br />
// flash of content<br />
this.custom(this.prop == "width" || this.prop == "height" ? 1 : 0, this.cur());<br />
<br />
// Start by showing the element<br />
jQuery(this.elem).show();<br />
},<br />
<br />
// Simple 'hide' function<br />
hide: function(){<br />
// Remember where we started, so that we can go back to it later<br />
this.options.orig[this.prop] = jQuery.style( this.elem, this.prop );<br />
this.options.hide = true;<br />
<br />
// Begin the animation<br />
this.custom(this.cur(), 0);<br />
},<br />
<br />
// Each step of an animation<br />
step: function(gotoEnd){<br />
var t = now();<br />
<br />
if ( gotoEnd || t >= this.options.duration + this.startTime ) {<br />
this.now = this.end;<br />
this.pos = this.state = 1;<br />
this.update();<br />
<br />
this.options.curAnim[ this.prop ] = true;<br />
<br />
var done = true;<br />
for ( var i in this.options.curAnim )<br />
if ( this.options.curAnim[i] !== true )<br />
done = false;<br />
<br />
if ( done ) {<br />
if ( this.options.display != null ) {<br />
// Reset the overflow<br />
this.elem.style.overflow = this.options.overflow;<br />
<br />
// Reset the display<br />
this.elem.style.display = this.options.display;<br />
if ( jQuery.css(this.elem, "display") == "none" )<br />
this.elem.style.display = "block";<br />
}<br />
<br />
// Hide the element if the "hide" operation was done<br />
if ( this.options.hide )<br />
jQuery(this.elem).hide();<br />
<br />
// Reset the properties, if the item has been hidden or shown<br />
if ( this.options.hide || this.options.show )<br />
for ( var p in this.options.curAnim )<br />
jQuery.style(this.elem, p, this.options.orig[p]);<br />
<br />
// Execute the complete function<br />
this.options.complete.call( this.elem );<br />
}<br />
<br />
return false;<br />
} else {<br />
var n = t - this.startTime;<br />
this.state = n / this.options.duration;<br />
<br />
// Perform the easing function, defaults to swing<br />
this.pos = jQuery.easing[this.options.easing || (jQuery.easing.swing ? "swing" : "linear")](this.state, n, 0, 1, this.options.duration);<br />
this.now = this.start + ((this.end - this.start) * this.pos);<br />
<br />
// Perform the next step of the animation<br />
this.update();<br />
}<br />
<br />
return true;<br />
}<br />
<br />
};<br />
<br />
jQuery.extend( jQuery.fx, {<br />
<br />
tick:function(){<br />
var timers = jQuery.timers;<br />
<br />
for ( var i = 0; i < timers.length; i++ )<br />
if ( !timers[i]() )<br />
timers.splice(i--, 1);<br />
<br />
if ( !timers.length )<br />
jQuery.fx.stop();<br />
},<br />
<br />
stop:function(){<br />
clearInterval( timerId );<br />
timerId = null;<br />
},<br />
<br />
speeds:{<br />
slow: 600,<br />
fast: 200,<br />
// Default speed<br />
_default: 400<br />
},<br />
<br />
step: {<br />
<br />
opacity: function(fx){<br />
jQuery.style(fx.elem, "opacity", fx.now);<br />
},<br />
<br />
_default: function(fx){<br />
if ( fx.elem.style && fx.elem.style[ fx.prop ] != null )<br />
fx.elem.style[ fx.prop ] = fx.now + fx.unit;<br />
else<br />
fx.elem[ fx.prop ] = fx.now;<br />
}<br />
}<br />
});<br />
if ( "getBoundingClientRect" in document.documentElement )<br />
jQuery.fn.offset = function() {<br />
var elem = this[0];<br />
if ( !elem || !elem.ownerDocument ) return null;<br />
if ( elem === elem.ownerDocument.body ) return jQuery.offset.bodyOffset( elem );<br />
var box = elem.getBoundingClientRect(), doc = elem.ownerDocument, body = doc.body, docElem = doc.documentElement,<br />
clientTop = docElem.clientTop || body.clientTop || 0, clientLeft = docElem.clientLeft || body.clientLeft || 0,<br />
top = box.top + (self.pageYOffset || jQuery.support.boxModel && docElem.scrollTop || body.scrollTop ) - clientTop,<br />
left = box.left + (self.pageXOffset || jQuery.support.boxModel && docElem.scrollLeft || body.scrollLeft) - clientLeft;<br />
return { top: top, left: left };<br />
};<br />
else<br />
jQuery.fn.offset = function() {<br />
var elem = this[0];<br />
if ( !elem || !elem.ownerDocument ) return null;<br />
if ( elem === elem.ownerDocument.body ) return jQuery.offset.bodyOffset( elem );<br />
jQuery.offset.initialize();<br />
<br />
var offsetParent = elem.offsetParent, prevOffsetParent = elem,<br />
doc = elem.ownerDocument, computedStyle, docElem = doc.documentElement,<br />
body = doc.body, defaultView = doc.defaultView,<br />
prevComputedStyle = defaultView.getComputedStyle(elem, null),<br />
top = elem.offsetTop, left = elem.offsetLeft;<br />
<br />
while ( (elem = elem.parentNode) && elem !== body && elem !== docElem ) {<br />
if ( jQuery.offset.supportsFixedPosition && prevComputedStyle.position === "fixed" ) break;<br />
computedStyle = defaultView.getComputedStyle(elem, null);<br />
top -= elem.scrollTop, left -= elem.scrollLeft;<br />
if ( elem === offsetParent ) {<br />
top += elem.offsetTop, left += elem.offsetLeft;<br />
if ( jQuery.offset.doesNotAddBorder && !(jQuery.offset.doesAddBorderForTableAndCells && /^t(able|d|h)$/i.test(elem.tagName)) )<br />
top += parseFloat( computedStyle.borderTopWidth ) || 0,<br />
left += parseFloat( computedStyle.borderLeftWidth ) || 0;<br />
prevOffsetParent = offsetParent, offsetParent = elem.offsetParent;<br />
}<br />
if ( jQuery.offset.subtractsBorderForOverflowNotVisible && computedStyle.overflow !== "visible" )<br />
top += parseFloat( computedStyle.borderTopWidth ) || 0,<br />
left += parseFloat( computedStyle.borderLeftWidth ) || 0;<br />
prevComputedStyle = computedStyle;<br />
}<br />
<br />
if ( prevComputedStyle.position === "relative" || prevComputedStyle.position === "static" )<br />
top += body.offsetTop,<br />
left += body.offsetLeft;<br />
<br />
if ( jQuery.offset.supportsFixedPosition && prevComputedStyle.position === "fixed" )<br />
top += Math.max( docElem.scrollTop, body.scrollTop ),<br />
left += Math.max( docElem.scrollLeft, body.scrollLeft );<br />
<br />
return { top: top, left: left };<br />
};<br />
<br />
jQuery.offset = {<br />
initialize: function() {<br />
var body = document.body, container = document.createElement('div'), innerDiv, checkDiv, table, td, bodyMarginTop = parseFloat( jQuery.curCSS(body, 'marginTop', true) ) || 0,<br />
html = '<div style="position:absolute;top:0;left:0;margin:0;border:5px solid #000;padding:0;width:1px;height:1px;"><div></div></div><table style="position:absolute;top:0;left:0;margin:0;border:5px solid #000;padding:0;width:1px;height:1px;" cellpadding="0" cellspacing="0"><tr><td></td></tr></table>';<br />
<br />
jQuery.extend( container.style, { position: 'absolute', top: 0, left: 0, margin: 0, border: 0, width: '1px', height: '1px', visibility: 'hidden' } );<br />
<br />
container.innerHTML = html;<br />
body.insertBefore( container, body.firstChild );<br />
innerDiv = container.firstChild, checkDiv = innerDiv.firstChild, td = innerDiv.nextSibling.firstChild.firstChild;<br />
<br />
this.doesNotAddBorder = (checkDiv.offsetTop !== 5);<br />
this.doesAddBorderForTableAndCells = (td.offsetTop === 5);<br />
<br />
checkDiv.style.position = 'fixed', checkDiv.style.top = '20px';<br />
this.supportsFixedPosition = (checkDiv.offsetTop === 20 || checkDiv.offsetTop === 15); // safari subtracts parent border width here which is 5px<br />
checkDiv.style.position = '', checkDiv.style.top = '';<br />
<br />
innerDiv.style.overflow = 'hidden', innerDiv.style.position = 'relative';<br />
this.subtractsBorderForOverflowNotVisible = (checkDiv.offsetTop === -5);<br />
<br />
this.doesNotIncludeMarginInBodyOffset = (body.offsetTop !== bodyMarginTop);<br />
<br />
body.removeChild( container );<br />
jQuery.offset.initialize = function(){};<br />
<br />
body = container = innerDiv = checkDiv = table = td = null;<br />
},<br />
<br />
bodyOffset: function(body) {<br />
jQuery.offset.initialize();<br />
var top = body.offsetTop, left = body.offsetLeft;<br />
if ( jQuery.offset.doesNotIncludeMarginInBodyOffset )<br />
top += parseFloat( jQuery.curCSS(body, 'marginTop', true) ) || 0,<br />
left += parseFloat( jQuery.curCSS(body, 'marginLeft', true) ) || 0;<br />
return { top: top, left: left };<br />
}<br />
};<br />
<br />
<br />
jQuery.fn.extend({<br />
position: function() {<br />
if ( !this[0] ) return null;<br />
<br />
var elem = this[0],<br />
<br />
// Get *real* offsetParent<br />
offsetParent = this.offsetParent(),<br />
<br />
// Get correct offsets<br />
offset = this.offset(),<br />
parentOffset = /^body|html$/i.test(offsetParent[0].tagName) ? { top: 0, left: 0 } : offsetParent.offset();<br />
<br />
// Subtract element margins<br />
// note: when an element has margin: auto the offsetLeft and marginLeft<br />
// are the same in Safari causing offset.left to incorrectly be 0<br />
offset.top -= parseFloat( jQuery.curCSS(elem, 'marginTop', true) ) || 0;<br />
offset.left -= parseFloat( jQuery.curCSS(elem, 'marginLeft', true) ) || 0;<br />
<br />
// Add offsetParent borders<br />
parentOffset.top += parseFloat( jQuery.curCSS(offsetParent[0], 'borderTopWidth', true) ) || 0;<br />
parentOffset.left += parseFloat( jQuery.curCSS(offsetParent[0], 'borderLeftWidth', true) ) || 0;<br />
<br />
// Subtract the two offsets<br />
return {<br />
top: offset.top - parentOffset.top,<br />
left: offset.left - parentOffset.left<br />
};<br />
},<br />
<br />
offsetParent: function() {<br />
var offsetParent = this[0].offsetParent || document.body;<br />
while ( offsetParent && (!/^body|html$/i.test(offsetParent.tagName) && jQuery.css(offsetParent, 'position') === 'static') )<br />
offsetParent = offsetParent.offsetParent;<br />
return jQuery( offsetParent );<br />
}<br />
});<br />
<br />
<br />
// Create scrollLeft and scrollTop methods<br />
jQuery.each( ['Left', 'Top'], function(i, name) {<br />
var method = 'scroll' + name;<br />
<br />
jQuery.fn[ method ] = function(val) {<br />
if ( !this[0] ) return null;<br />
<br />
var elem = this[0], win = ("scrollTo" in elem && elem.document) ? elem :<br />
(elem.nodeName === "#document") ? elem.defaultView || elem.parentWindow :<br />
false;<br />
<br />
return val !== undefined ?<br />
<br />
// Set the scroll offset<br />
this.each(function() {<br />
win = ("scrollTo" in this && this.document) ? this : <br />
(this.nodeName === "#document") ? this.defaultView || this.parentWindow :<br />
false;<br />
<br />
win ?<br />
win.scrollTo(<br />
!i ? val : jQuery(win).scrollLeft(),<br />
i ? val : jQuery(win).scrollTop()<br />
) :<br />
this[ method ] = val;<br />
}) :<br />
<br />
// Return the scroll offset<br />
win ?<br />
win[ i ? 'pageYOffset' : 'pageXOffset' ] ||<br />
jQuery.support.boxModel && win.document.documentElement[ method ] ||<br />
win.document.body[ method ] :<br />
elem[ method ];<br />
};<br />
});<br />
// Create innerHeight, innerWidth, outerHeight and outerWidth methods<br />
jQuery.each([ "Height", "Width" ], function(i, name){<br />
<br />
var type = name.toLowerCase();<br />
<br />
// innerHeight and innerWidth<br />
jQuery.fn["inner" + name] = function(){<br />
return this[0] ?<br />
jQuery.css( this[0], type, false, "padding" ) :<br />
null;<br />
};<br />
<br />
// outerHeight and outerWidth<br />
jQuery.fn["outer" + name] = function(margin) {<br />
return this[0] ?<br />
jQuery.css( this[0], type, false, margin ? "margin" : "border" ) :<br />
null;<br />
};<br />
<br />
jQuery.fn[ type ] = function( size ) {<br />
// Get window width or height<br />
var elem = this[0];<br />
if ( !elem ) return null;<br />
return ("scrollTo" in elem && elem.document) ? // does it walk and quack like a window?<br />
// Everyone else use document.documentElement or document.body depending on Quirks vs Standards mode<br />
elem.document.compatMode === "CSS1Compat" && elem.document.documentElement[ "client" + name ] ||<br />
elem.document.body[ "client" + name ] :<br />
<br />
// Get document width or height<br />
(elem.nodeName === "#document") ? // is it a document<br />
// Either scroll[Width/Height] or offset[Width/Height], whichever is greater<br />
Math.max(<br />
elem.documentElement["client" + name],<br />
elem.body["scroll" + name], elem.documentElement["scroll" + name],<br />
elem.body["offset" + name], elem.documentElement["offset" + name]<br />
) :<br />
<br />
// Get or set width or height on the element<br />
size === undefined ?<br />
// Get width or height on the element<br />
jQuery.css( elem, type ) :<br />
<br />
// Set the width or height on the element (default to pixels if value is unitless)<br />
this.css( type, typeof size === "string" ? size : size + "px" );<br />
};<br />
<br />
});<br />
})(window);</div>Frans