Team:Groningen/Project

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(Final products:: Added table summarizing importers, accumulators and promoters (unfinished).)
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<h1>Heavy metal scavengers with a vertical gas drive</h1>
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'''Introduction:'''
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==For our team==
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Human health and the environment are endangered by heavy metal pollution in water and sediment.  To improve purification strategies a  metal selective microbacterial cleaning system was designed.  The system comprises uptake, sequestering and  metal sensitive buoyancy. 
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{| border="1"
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All subsystems are interchangeable, which makes it suitable for almost any metal cleaning assay.  For this project the modular system was focused on arsenic accumulation, using ''Escherichia coli'' as a chassis organism.
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|+'''Missing / available information'''
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Arsenite  and arsenate are imported  by GlpF, a aquaglycerol porin from ''E. coli''. Intracellular As(III) and As(V) are sequestered by fMT or ArsR. These proteins were used as the accumulation modules. Since ''E. coli'' does not have a buoyancy system, the polycistronic  gas vesicle protein gene cluster from ''Bacillus megaterium'', GVP, was used. The arsenic promoter from ''E. coli'', pArsR, is regulated by the negative transcriptional regulator ArsR. GVP, under  regulation of pArsR, was used as the metal sensitive buoyancy module.
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!Metal
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</div>
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!Transporter
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|style="vertical-align:top;width:583px;"|
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!Inducible promoter
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{{imageMap|imap|/wiki/images/8/89/Arsenic_Filtering_System.png|610|410|
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!Regulator
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{{imageMapLink|arsenic|35|0|149|77|/wiki/images/2/2d/Arsenic_Filtering_System_-_Arsenic.png}}
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!Accumulation protein
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{{imageMapLink|transport|51|55|137|120|/wiki/images/4/44/Arsenic_Filtering_System_-_Transport.png}}
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|-
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{{imageMapLink|accumulation|183|143|153|77|/wiki/images/8/84/Arsenic_Filtering_System_-_Accumulation.png}}
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|Arsenic
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{{imageMapLink|gas|401|86|137|106|/wiki/images/7/72/Arsenic_Filtering_System_-_Gas_Vesicles.png}}
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|GlpF (organism?) - ordered
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{{imageMapLink|promoter|114|295|168|63|/wiki/images/5/5c/Arsenic_Filtering_System_-_Promoter.png}}
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|style="color:orange;"|Promoter region of ?? gene, responding on ArsR
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}}<div style="display:block;position:relative;float:right;">
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|ArsR (''E. coli'')- ordered
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<div id="imap" style="position:absolute;right:0px;"><div style="position:absolute;bottom:31px;right:72px;"></div>
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|ArsR  
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<html><ul>
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|-
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<li id="arsenic"></html>It all starts with [[Team:Groningen/Application|arsenic]] in solution.<html></li>
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|Copper
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<li id="transport"></html>[[Team:Groningen/Project/Transport|Metal transport]]<html></li>
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|HmtA (''Pseudomonas'' sp.)- available
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<li id="accumulation"></html>[[Team:Groningen/Project/Accumulation|Metal accumulation]]<html></li>
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|style="color:red;"|None found in ''E. coli''
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<li id="gas"></html>[[Team:Groningen/Project/Vesicle|Gas Vesicle]]<html></li>
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|style="color:red;"|Idem
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<li id="promoter"></html>[[Team:Groningen/Project/Promoters|Metal sensitive promoters]]<html></li>
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|style="color:red;"|??
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</ul></html>
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|-
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</div></div>
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|Zinc
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|HmtA (''Pseudomonas'' sp.)- available
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|style="color:red;"|??
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|style="color:red;"|??
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|style="color:red;"|??
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|-
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|Mercury
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|MerT (''E. coli'' and other sp)- PCR?
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|style="color:orange;"|Should be available in ''E. coli'' - PCR?
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|style="color:red;"|Idem
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|style="color:red;"|Idem
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|}
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.intro h1 { font-weight:bold; font-size:medium; border:none; margin:0px !important; padding:0px; }
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<div style="float:right;width:0px;height:150px;"></div>
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'''Results:'''
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==Introduction==
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All modules were cloned according to the BioBrick<sup>TM</sup>  Standard Assembly 10. The synthetic gene GlpF ,  was successfully cloned into a synthetic operon, with fMT. The GVP cluster, with a ten times repeat sequence, was successfully cloned downstream of the pArsR promoter. These two subsystems were transformed in  ''E. coli'' to complete the system.
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For the lab work of our project, concerning the bouyant bacteria with its metal absorbing and accumulating "function", we hope to produce the following products by using our basic cloning strategy.
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The system and its subparts were tested using several assays. Accumulation was tested by an uptake assay, however, since no reproducible results were obtained, the functionality of the accumulation module could not be determined from these data. Arsenic uptake was examined using a metal sensitivity assay. The ''E. coli'' strain overexpressing GlpF showed a decreased final cell density upon induction with As(III), suggesting functional expression of the transporter. The metal sensitive promoter pArsR  was tested using a fluorescence assay. This showed a 2.3 fold increased activity upon induction with 100 µM NaAsO<sub>2</sub>.
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Buoyancy was tested by a sedimentation assay.  Enhanced buoyancy was shown for the buoyancy module and the complete system, though no difference of the buoyancy phenotype could be observed upon addition of the accumulation module. Cells cultivated in aerobic conditions showed improved buoyancy compared to cells cultivated in semi-aerobic conditions. Expression of gas vescicles was shown by electron microscopy.
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An interactive computer model was made for the whole system, with which the modules were further characterized. With the model, import rates of As(III) at different initial extracellular arsenic concentrations could be determined. Also the influence of different parameters on the accumulation factor, the ratio between bound and unbound arsenic, was calculated.  The model also allowed qualitative determination of the regulation of pArsR by various expression levels of ArsR. Furthermore, the volume fraction gas vesicles in the cells needed for buoyancy, for several sizes of the gas vesicles, was computed.
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==Final products:==
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'''Conclusion:'''
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*Plasmid with gvp cluster, regulated by different promoters. [link to BioBricks]
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* (Several) plasmid(s) with a metal transporter, a metal accumulating protein and if needed a regulator protein for the metal sensitive promoter. [link to BioBricks]
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*One ''E. coli'' strain with both systems.
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[https://2009.igem.org/Team:Groningen/Parts Part List] for our -80 freezer!
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The metal selective microbacterial cleaning system for arsenic was shown to be buoyant and the buoyancy module and uptake module were shown to work individually. For a better determination of the system an accumulation assay need to be redone. It was shown here that the system has potential as a cleaning system for arsenic.
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As mentioned earlier this modular system can also be implemented in cleaning of other substances. Literature research showed possible modules for copper, zinc, mercury and even gold. So not only cleaning water and sludge but also mining rare metals could be functionalized using this system.
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<br><br><br>
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</div>
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In the periodic table below you can see for which elements we have identified a transporter, an accumulating protein and/or promotor. {{todo}} Make list more complete and add links to parts.
 
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{{periodic_table
 
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|AsInfo=
 
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Transporter: GlpF<br/>
 
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Accumulator: ArsR<br/>
 
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|SbInfo=
 
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Transporter: GlpF<br/>
 
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|CuInfo=
 
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Transporter: {{part|BBa_K190018|HmtA}}<br/>
 
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Accumulator: SmtA<br/>
 
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|ZnInfo=
 
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Transporter: {{part|BBa_K190018|HmtA}}<br/>
 
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Accumulator: SmtA<br/>
 
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|HgInfo=
 
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Transporter: MerT<br/>
 
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Accumulator: SmtA<br/>
 
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|CdInfo=
 
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Accumulator: SmtA<br/>
 
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}}
 
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Alternatively this information can be summarized in the following table:
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{{Team:Groningen/Footer}}
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{|border="1"
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!
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!colspan="2"|Importers
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!colspan="3"|Accumulators
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!Promoters
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|-
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!
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!GlpF
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!{{part|BBa_K190018|HmtA}}
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!ArsR
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!SmtA
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!MymT
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|-
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!Cu
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|-
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!Zn
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|-
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!As
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|-
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!Cd
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|-
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!Sb
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|-
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!Hg
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|}
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==Order of action:==
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# Buoyancy
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# Metal importation
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# Accumulation
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# Metal sensitive promotor
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==Basic Cloning Strategy:==
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# Transform ''E. coli'' TOP10 (genotype DH 10B) with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins.
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# PCR the restriction sites out and add BioBrick pre- and suffix --> Use [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC10 BBFRCF10].
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##Primers should be ordered for the different genes.
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## Add a RBS (BBa_B0034)in the primer for the BioBrick prefix.
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## Add a terminator (BBa_B0014) via cloning.
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## For gvp the RBS is included in the construct, and biobrick suffix is included in the construct. The prefix is missing because of the ''Eco''RI site in the middle of the plasmid!! This may give problems!!
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# PCR restriction sites out. '''!!Both PCR reactions for pre/suffix and restriction sites can possibly be done in 3 PCR reactions --> Ask Frans or J. Kok!!'''
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# Test expression / phenotype '''of separate proteins''' (if possible in the vectors they are supplied in).
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# Put both systems (gvp and metal import) on a high and low copy number (supplied by "vector group"). This is needed to prevent plasmid / expression incompatibility when both systems are used in one strain.
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## The metal transporter and accumulation protein should be cloned behind each other. If possible on a synthetic operon.
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## Clone the different systems for Cu, Zn, As, (Hg) in [http://www.partsregistry.org/Assembly:Rolling_assembly  parallel].
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# ''(If needed and not already supplied by "vector group")'' [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.
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# ''(If needed and not already supplied by "vector group")'' [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] different promoters in front of the two systems.
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## Inducible like Para or Plac
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## Constitutive with expected high and low expression yield
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## Metal sentitive promoter (only for gvp system)
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# Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors
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-
 
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==Quality control==
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What to do after transforming your cells with a vector:
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*Grow o/n culture as usual
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*Streak with "entoog" a little out for single colonies.
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*Thereafter you can continue to miniprep the cells and check insert length by PCR/restriction analysis (Quality control --> Overdrachts document iGEM 2008).
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** For restriction analysis, do a double digest, and try to find a restriction enzyme which only cuts in the vector and one which only cuts in the insert...
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*Pick one (very) single colony, try hard not to pick a satellite colony!
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*Grow o/n culture and make glycerol stock (keep track of were you put them in "constructs" file on Google Docs!) next day.
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*Miniprep and continue with cloning / checking insert length.
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-
 
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==Tracking information about cloning==
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*Constructs / plasmids lokation in GoogleDocs document called: '''Constructs'''.
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*Primer / Oligos in GoogleDocs documents called: '''Primers'''.
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*What we take from MolGen stocks in a GoogleDocs document called: '''Chemicals / Materials taken from MolGen'''.
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**This list was also put on the door in the lab.
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-
 
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[https://2009.igem.org/Team:Groningen/Parts Part List] for our -80 freezer!
+

Latest revision as of 20:55, 21 October 2009

Igemhomelogo.png
The Project
GroningenPrevious.png
Next.JPG

Heavy metal scavengers with a vertical gas drive

Introduction:

Human health and the environment are endangered by heavy metal pollution in water and sediment. To improve purification strategies a metal selective microbacterial cleaning system was designed. The system comprises uptake, sequestering and metal sensitive buoyancy. All subsystems are interchangeable, which makes it suitable for almost any metal cleaning assay. For this project the modular system was focused on arsenic accumulation, using Escherichia coli as a chassis organism. Arsenite and arsenate are imported by GlpF, a aquaglycerol porin from E. coli. Intracellular As(III) and As(V) are sequestered by fMT or ArsR. These proteins were used as the accumulation modules. Since E. coli does not have a buoyancy system, the polycistronic gas vesicle protein gene cluster from Bacillus megaterium, GVP, was used. The arsenic promoter from E. coli, pArsR, is regulated by the negative transcriptional regulator ArsR. GVP, under regulation of pArsR, was used as the metal sensitive buoyancy module.

Results:

All modules were cloned according to the BioBrickTM Standard Assembly 10. The synthetic gene GlpF , was successfully cloned into a synthetic operon, with fMT. The GVP cluster, with a ten times repeat sequence, was successfully cloned downstream of the pArsR promoter. These two subsystems were transformed in E. coli to complete the system. The system and its subparts were tested using several assays. Accumulation was tested by an uptake assay, however, since no reproducible results were obtained, the functionality of the accumulation module could not be determined from these data. Arsenic uptake was examined using a metal sensitivity assay. The E. coli strain overexpressing GlpF showed a decreased final cell density upon induction with As(III), suggesting functional expression of the transporter. The metal sensitive promoter pArsR was tested using a fluorescence assay. This showed a 2.3 fold increased activity upon induction with 100 µM NaAsO2. Buoyancy was tested by a sedimentation assay. Enhanced buoyancy was shown for the buoyancy module and the complete system, though no difference of the buoyancy phenotype could be observed upon addition of the accumulation module. Cells cultivated in aerobic conditions showed improved buoyancy compared to cells cultivated in semi-aerobic conditions. Expression of gas vescicles was shown by electron microscopy. An interactive computer model was made for the whole system, with which the modules were further characterized. With the model, import rates of As(III) at different initial extracellular arsenic concentrations could be determined. Also the influence of different parameters on the accumulation factor, the ratio between bound and unbound arsenic, was calculated. The model also allowed qualitative determination of the regulation of pArsR by various expression levels of ArsR. Furthermore, the volume fraction gas vesicles in the cells needed for buoyancy, for several sizes of the gas vesicles, was computed.

Conclusion:

The metal selective microbacterial cleaning system for arsenic was shown to be buoyant and the buoyancy module and uptake module were shown to work individually. For a better determination of the system an accumulation assay need to be redone. It was shown here that the system has potential as a cleaning system for arsenic. As mentioned earlier this modular system can also be implemented in cleaning of other substances. Literature research showed possible modules for copper, zinc, mercury and even gold. So not only cleaning water and sludge but also mining rare metals could be functionalized using this system.