http://2009.igem.org/wiki/index.php?title=Special:Contributions/Evangelineoh&feed=atom&limit=50&target=Evangelineoh&year=&month=2009.igem.org - User contributions [en]2024-03-29T09:38:48ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:EdinburghTeam:Edinburgh2009-10-21T18:45:25Z<p>Evangelineoh: </p>
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<div id="references"><br />
[1] Landmine monitor 2009. <font color="#932a0e">Landmine Monitor Fact Sheet. Landmines and children.</font><br />
<a href="http://www.lm.icbl.org/index.php/content/view/full/24160">http://www.lm.icbl.org/index.php/content/view/full/24160</a><br />
<br /><br /><br />
[2] Thomas F. Jenkins, Daniel C. Leggett, Paul H. Miyares, Marianne E. Walsh, Thomas A. Ranney, James H. Cragin and Vivian George. 2001. <font color="#932a0e">Chemical signatures of TNT-filled land mines.</font> <i>Talanta.</i> <font color="#932a0e">54</font> (3):501-513 <br />
<br /><br /><br />
[3] Christopher E. French, Stephen Nicklin, and Neil C. Bruce. 1998 <font color="#932a0e">Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase.</font> <i>Applied and Environmental Microbiology</i>, <font color="#932a0e">64</font> (8): 2864-2868<br />
<br /><br /><br />
[4] Seth-Smith, H.M.B., Rosser, S.J., Basran, A., Travis, E.R., Dabbs, E.R., Nicklin, S. andBruce, N.C. 2002. <font color="#932a0e">Cloning, Sequencing and Characterization of the Hexahydro-1,3,5-trinitro-1,3,5-triazine Degradation Gene Cluster from <i>Rhodococcus rhodochrous</i>.</font> <i>Appl. Environ. Microbiol.</i> <font color="#932a0e">68</font>:4764-4771.<br />
<br /><br /><br />
[5] Smith, C.J. & Chalk, P.M. 1980. <font color="#932a0e">Gaseous nitrogen evolution during nitrification of ammonia fertilizer and nitrite transformation in soils.</font> <i>Soil Science Society of America Journal</i>, <font color="#932a0e">44</font>, 277–282.<br />
<br /><br /><br />
[6] Burns, L.C., Stevens, R.J. & Laughlin, R.J. 1995. <font color="#932a0e">Determination of the simultaneous production and consumption of soil nitrite using 15N.</font> <i>Soil Biology and Biochemistry</i>, <font color="#932a0e">27</font>, 839–844.<br />
<br /><br /><br />
[7] Van Cleemput, O. & Samater, A.H. 1996. <font color="#932a0e">Nitrite in soils: accumulation and role in the formation of gaseous N compounds.</font> <i>Fertilizer Research</i>, <font color="#932a0e">45</font>, 81–89.<br />
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<div id="references2"><br />
<br />
Burlage, R., M. Hunt, J. DiBenedetto, and M. Maston. <font color="#932a0e">Locating TNT with BioReporter Bacteria.</font> <i>The University of Western Australia.</i> Web. 12 Oct. 2009. <a href="http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html">http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html</a><br />
<br /><br /><br />
<font color="#932a0e">Method for detection of buried explosives using a biosensor - US Patent 5972638 Abstract.</font> <i>PatentStorm: U.S. Patents.</i> Web. 12 Oct. 2009. <a href="http://www.patentstorm.us/patents/5972638.html">http://www.patentstorm.us/patents/5972638.html</a><br />
<br /><br /><br />
<font color="#932a0e">Reducing the Threat of War and Terrorism.</font> <i>Oak Ridge National Laboratory.</i> Web. 12 Oct. 2009. <a href="http://www.ornl.gov/info/ornlreview/meas_tech/threat.htm">http://www.ornl.gov/info/ornlreview/meas_tech/threat.htm</a><br />
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<br /><br />
<b>iGEM PROJECT "TNT/RDX Biosensor and Bioremediator" </a></b><br />
<br /><br /><br />
In 2007, <b>5 426</b> new casualties were recorded from <a class="load-local" href="#landmines" rel="#landmines">landmine</a> explosions. 71% of these casualties were civilians. A further 46% of the civilian casualties were children<sup><a class="load-local" href="#references" rel="#references">[1]</a></sup>. This made us realise the need for the production of a cheap, safe and accurate method that can be applied in a big scale to help detect landmines. A synthetic organism could be just what is needed.<br />
<br /><br /><br />
Even though landmines are buried under soil, there is leakage which indicates their imminent position with a chemical fingerprint. TNT-filled landmines produce three major source chemicals, namely 1,3-DNB, 2,4-DNT, and 2,4,6-TNT <sup><a class="load-local" href="#references" rel="#references">[2]</a></sup>. In addition, the natural degradation of explosive compounds, such as TNT, by bacterial enzymes produces nitrogen in the form of Nitrites<sup><a class="load-local" href="#references" rel="#references">[3]</a></sup>. Nitrites are also one of the by-products of the degradation of another explosive used in landmines, namely RDX. In the latter case, this can be achieved by the soil bacterium <i>Rhodococcus rhodochrous</i><sup><a class="load-local" href="#references" rel="#references">[4]</a></sup>.<br />
<br /><br /><br />
<b>Our project is concerned in making a biosensor that would detect both the presence of TNT and nitrites/nitrates.</b> <br />
<br /><br /><br />
Natural nitrite concentration in soil tends to be very low (below 0.1 mg NO2-N /kg)<sup><a class="load-local" href="#references" rel="#references">[7]</a></sup>. Thereby the possibility of false positive results decreases. Our biosensor would also detect nitrates but these would need to be at a much higher concentration than nitrites to bring about a response.<br />
<br /><br /><br />
The fact that excessive fertilisation with ammonium producing fertilizers such as urea can cause an increase in the presence of nitrites in the soil <sup><a class="load-local" href="#references" rel="#references">[5]</a></sup><sup><a class="load-local" href="#references" rel="#references">[6]</a></sup><sup><a class="load-local" href="#references" rel="#references">[7]</a></sup> gives the possibility that our device can be used in diverse fields of interest, from landmine identification (ranging from TNT landmines to RDX ones), to assaying extent of fertiliser induced nitrite/nitrate pollution. <br />
<br /><br /><br />
Please read our detailed project description and part characterisation for further details. You might also want to visit our <a href="https://2009.igem.org/Team:Edinburgh/projectmain/motivation">motivation page</a> to see why our project might help lots of people.<br />
<br />
<br /><br /><br />
<br />
<div id="r"><br />
<b>References</b><br />
<br /><br /><br />
[1] Landmine monitor 2009. <b>Landmine Monitor Fact Sheet. Landmines and children.</b><br />
<a href="http://www.lm.icbl.org/index.php/content/view/full/24160">http://www.lm.icbl.org/index.php/content/view/full/24160</a><br />
<br /><br /><br />
[2] Thomas F. Jenkins, Daniel C. Leggett, Paul H. Miyares, Marianne E. Walsh, Thomas A. Ranney, James H. Cragin and Vivian George. 2001. <b>Chemical signatures of TNT-filled land mines.</b> <i>Talanta.</i> <b>54</b> (3):501-513 <br />
<br /><br /><br />
[3] Christopher E. French, Stephen Nicklin, and Neil C. Bruce. 1998 <b>Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase.</b> <i>Applied and Environmental Microbiology</i>, <b>64</b> (8): 2864-2868<br />
<br /><br /><br />
[4] Seth-Smith, H.M.B., Rosser, S.J., Basran, A., Travis, E.R., Dabbs, E.R., Nicklin, S. andBruce, N.C. 2002. <b>Cloning, Sequencing and Characterization of the Hexahydro-1,3,5-trinitro-1,3,5-triazine Degradation Gene Cluster from <i>Rhodococcus rhodochrous</i>.</b> <i>Appl. Environ. Microbiol.</i> <b>68</b>:4764-4771.<br />
<br /><br /><br />
[5] Smith, C.J. & Chalk, P.M. 1980. <b>Gaseous nitrogen evolution during nitrification of ammonia fertilizer and nitrite transformation in soils.</b> <i>Soil Science Society of America Journal</i>, <b>44</b>, 277–282.<br />
<br /><br /><br />
[6] Burns, L.C., Stevens, R.J. & Laughlin, R.J. 1995. <b>Determination of the simultaneous production and consumption of soil nitrite using 15N.</b> <i>Soil Biology and Biochemistry</i>, <b>27</b>, 839–844.<br />
<br /><br /><br />
[7] Van Cleemput, O. & Samater, A.H. 1996. <b>Nitrite in soils: accumulation and role in the formation of gaseous N compounds.</b> <i>Fertilizer Research</i>, <b>45</b>, 81–89.<br />
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<b>Why we differ<a name="why">?</a></b><br />
<br /><br /><br />
<b>Existing systems</b><br />
<br /><br /><br />
Biological systems for mine and particularly TNT detection have been described previously, the most prominent of which is the patented Microbial Mine Detection System (MMDS) developed by Dr Paul Burlage and the Oak Ridge National Laboratory. The MMDS is normally a bacterium able to detect and exhibit chemotaxis towards TNT and the vapours released as a result of its degradation, in particular nitrates and nitrites. The bacteria utilized at the Oak Ridge Laboratory are mainly the <i>Bacillus</i> or <i>Pseudomonas</i> species, such as <i>Pseudomonas putida</i>, which have been found to naturally exhibit growth towards a source of TNT degradation. Although the MMDS patent states that the recombinant microorganism is able to directly detect TNT, it is likely that the bacteria do not exhibit chemotaxis towards TNT per se but rather the organism is sensitive to high levels of nitrate and nitrite. The sensitive genes are in turn fused with a Green Fluorescent Protein (GFP) reporter gene, making it possible to visualize the fluorescent bacteria with the help of a UV illuminator. The MMDS has been field tested at a South Carolina site, where the system was able to locate all five of the hidden mine sites, showing the great potential of biological mine detection (Fig. 1). <br />
<br />
<br /><br /><br />
<center><img src="https://static.igem.org/mediawiki/2009/0/05/WhyWeDifferFig1.jpg" width="500"></center><br />
<center><i>Figure 1. An image showing a UV scan of a 3x4 metre field test area, demonstrating higher fluorescence levels around the mine site as well as plant material, possibly due to TNT being absorbed through the root system of surrounding plants. <a class="title" href="#" title="|Image from Burlage <i>et al</i>, 1999<br />
<a href='http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html' target='_blank'>http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html</a>|">Image source</a></sup></i></center><br />
<br />
<br /><br /><br />
<br />
<b>Advantages of our system</b><br />
<br />
<br /><br /><br />
<br />
The primary advantage of our system over the MMDS is the specificity of the TNT detection system. Beside the nitrate and nitrite sensitivity, the MineBusters system (MBS) is able to exhibit high specificity to TNT with the help of a completely novel computationally designed TNT-specific promoter. Prior to this creation TNT specificity has not been found to occur in nature. Due to this the MBS is able to provide much more accurate readings of mine presence. With the help of the enzyme nitroreductase the MBS is also able to degrade TNT, providing further signal for the system. Most importantly, the MBS signals the presence of TNT as a combination of light and fluorescence-emitting systems, providing a <b>directly visible</b> outcome of various colours and in this way eliminating the need for any scanning or illumination equipment. We believe that on the basis of this our system has the potential to be much more accurate, easier and cheaper to operate than the existing one. We are confident that our system is a great candidate for a cheap, accessible and easy to use mine detection and eradication method.<a class="load-local" href="#references2" rel="#references2"></a><br />
<br />
<div id="r"><br />
<br /><br /><br />
<b>References</b><br />
<br /><br /><br />
Burlage, R., M. Hunt, J. DiBenedetto, and M. Maston. <b>Locating TNT with BioReporter Bacteria.</b> <i>The University of Western Australia.</i> Web. 12 Oct. 2009. <a href="http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html">http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html</a><br />
<br /><br /><br />
<b>Method for detection of buried explosives using a biosensor - US Patent 5972638 Abstract.</b> <i>PatentStorm: U.S. Patents.</i> Web. 12 Oct. 2009. <a href="http://www.patentstorm.us/patents/5972638.html">http://www.patentstorm.us/patents/5972638.html</a><br />
<br /><br /><br />
<b>Reducing the Threat of War and Terrorism.</b> <i>Oak Ridge National Laboratory.</i> Web. 12 Oct. 2009. <a href="http://www.ornl.gov/info/ornlreview/meas_tech/threat.htm">http://www.ornl.gov/info/ornlreview/meas_tech/threat.htm</a><br />
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<div id="Edinburghfooter"><center><font color="#ffffff"><div style="margin-top:40px;">iGEM Team Edinburgh 2009 sends a huge <font color="red">THANK YOU</font> to our sponsors.<br />
<br /><br /><br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T15:43:05Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Blog Entry</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
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<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">tnt.r1-trz</a> (BBa_K216013) <br /><br />
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</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
</div><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J58104">(BBa_J58104)</a>. However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trz, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">BBa_K216004</a>) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a>. The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by <i>luxCDE</i> (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens<br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216008">(BBa_K216008)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>We worked extremely hard to clone <i>luxAB</i> from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned <i>luxAB</i> from Xenorhabdus luminescens. This was successful- hosts expressing <i>luxAB</i> (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal).<br />
<br><br />
<br><br />
<img src="http://partsregistry.org/wiki/images/8/80/XL_luxAB.jpg"><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of <i>tnt.r1</i>, <i>trz</i> and <i>luxCDE</i> genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PompC <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a> and <i>lacZ'</i> <a href="http://partsregistry.org/Part:BBa_J33202">(BBa_J33202)</a>. Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>lacZ'’</i> <a href="http://partsregistry.org/Part:BBa_J33202">(Bba_J33202)</a>. Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>Renilla luciferase</i> <a href="http://partsregistry.org/Part:BBa_J52008">(BBa_J52008)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_J52008">BBa_J52008</a> from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and firefly luciferase <a href="http://partsregistry.org/Part:BBa_I712019">(BBa_I712019)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_I712019">BBa_I712019</a> from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> <a href="http://partsregistry.org/Part:BBa_K216002">(BBa_ K216002)</a> and <i>trz</i> <a href="http://partsregistry.org/Part:BBa_K216004">(BBa_ K216004)</a>.</td><br />
<br />
</tr><br />
<br />
</table><br />
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</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(results)Team:Edinburgh/biology(results)2009-10-21T15:38:57Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Results</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<b> Personal Note </b> <br /><br /><br />
One of the best things about iGEM is that you get to learn <b>a lot</b> from your peers. Biologists learn about modelling, engineers learn about biology...and we all have to learn a little <i>HTML</i>. The learning can be intensely stressful at times but also very satisfying.<br />
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<font color="green" style="float:right">Evangeline</font><br />
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What is even more important, we learn from each other not just the technical knowledge, but something that is more precious to me - life views. Learning from others life views, you can extend and adjust your personal life, make it bigger, smarter and more adaptive.<br />
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<font color="green" style="float:right">JD</font><br />
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<img src="https://static.igem.org/mediawiki/2009/7/71/OmpcCharachterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/1/13/Ompc-promoter.jpg"> </center><br />
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We used Jason Kelly's promoter characterization kit to characterize the <i>ompC</i> promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.<br />
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<img src="https://static.igem.org/mediawiki/2009/b/bb/YeaRPRomoterCharacterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<b> Miller Test </b> <br /><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/e/ef/Millertest.jpg"> </center> <br /><br /><br />
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The classical Miller's Assay was used to characterize the <i>yeaR</i> promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates and nitrites, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.<br />
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<b>PyeaR characterization with fluorescence using Jason Kelly's kit</b> <br /><br />
<u>PyeaR response to nitrates</u><br />
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Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrates. The most sensitive time period is between 5 ½ hours (320 minutes) 7 ½ hours (440 minutes) after induction. The sensitivity decreases with time, but induction is still at a high level after the peak activity of the promoter. Only two concentrations are shown (0 mM and 10 mM), as we have demonstrated that 10 mM is the sensitivity peak of this promoter (look at last graph). <br />
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<u>PyeaR response to nitrites</u><br />
<br><br />
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrites. The fluorescence levels begin to build up only after about 8 hours; therefore other irrelevant results are omitted from this graph. The sensitivity curve in this case is less steep compared to the one produced due to the presence of nitrates. This indicates that the promoter responds differently to these two nitrogenous compounds. Again, only two concentrations are shown (0 mM and 10 mM), as sensitivity peaks at 10mM.<br />
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<u>PyeaR response to nitrates and nitrites after overnight induction</u><br />
<br><br />
Here we can compare the activity of this promoter to different concentrations of both nitrates and nitrates. We observed that the promoter is activated more readily by nitrates compared to nitrites. Nevertheless, the activation produced by nitrites remains significant. The peak activation concentration for both compounds is 10 mM, after which activation efficiency decreases.<br />
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<center> <img src="https://static.igem.org/mediawiki/2009/e/e7/EdinburghPyear_characterization.jpg"> </center> <br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8d/EdinburghFurtherWork.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed! <br />
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<b>Testing TNT receptors</b><br /><br /><br />
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First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.<br />
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<b>Environmental aspect</b> <br /><br /><br />
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For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.<br />
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<b>Field test</b> <br /><br /><br />
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Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient. <br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(results)Team:Edinburgh/biology(results)2009-10-21T15:37:07Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Results</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:220px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal Note </b> <br /><br /><br />
One of the best things about iGEM is that you get to learn <b>a lot</b> from your peers. Biologists learn about modelling, engineers learn about biology...and we all have to learn a little <i>HTML</i>. The learning can be intensely stressful at times but also very satisfying.<br />
<br /><br /><br />
<font color="green" style="float:right">Evangeline</font><br />
<br />
<br /><br /><br />
What is even more important, we learn from each other not just the technical knowledge, but something that is more precious to me - life views. Learning from others life views, you can extend and adjust your personal life, make it bigger, smarter and more adaptive.<br />
<br /><br /><br />
<font color="green" style="float:right">JD</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/7/71/OmpcCharachterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/1/13/Ompc-promoter.jpg"> </center><br />
<br />
<br /><br /><br />
<br />
We used Jason Kelly's promoter characterization kit to characterize the <i>ompC</i> promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.<br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/b/bb/YeaRPRomoterCharacterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<br /><br />
<b> Miller Test </b> <br /><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2009/e/ef/Millertest.jpg"> </center> <br /><br /><br />
<br />
The classical Miller's Assay was used to characterize the <i>yeaR</i> promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates and nitrites, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.<br />
<br />
<br /><br /><br />
<br />
<b>PyeaR characterization with fluorescence using Jason Kelly's kit</b> <br /><br />
<br><br />
<i>Data interpretation in text after graphs.</i><br />
<br><br />
<center> <img src="https://static.igem.org/mediawiki/2009/e/e7/EdinburghPyear_characterization.jpg"> </center> <br /><br /><br />
<br><br />
<br><br />
<u>PyeaR response to nitrates</u><br />
<br><br />
<br />
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrates. The most sensitive time period is between 5 ½ hours (320 minutes) 7 ½ hours (440 minutes) after induction. The sensitivity decreases with time, but induction is still at a high level after the peak activity of the promoter. Only two concentrations are shown (0 mM and 10 mM), as we have demonstrated that 10 mM is the sensitivity peak of this promoter (look at last graph). <br />
<br><br><br />
<u>PyeaR response to nitrites</u><br />
<br><br />
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrites. The fluorescence levels begin to build up only after about 8 hours; therefore other irrelevant results are omitted from this graph. The sensitivity curve in this case is less steep compared to the one produced due to the presence of nitrates. This indicates that the promoter responds differently to these two nitrogenous compounds. Again, only two concentrations are shown (0 mM and 10 mM), as sensitivity peaks at 10mM.<br />
<br><br><br />
<u>PyeaR response to nitrates and nitrites after overnight induction</u><br />
<br><br />
Here we can compare the activity of this promoter to different concentrations of both nitrates and nitrates. We observed that the promoter is activated more readily by nitrates compared to nitrites. Nevertheless, the activation produced by nitrites remains significant. The peak activation concentration for both compounds is 10 mM, after which activation efficiency decreases.<br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/8/8d/EdinburghFurtherWork.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed! <br />
<br />
<br /><br /><br />
<br />
<b>Testing TNT receptors</b><br /><br /><br />
<br />
First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.<br />
<br />
<br /><br /><br />
<br />
<b>Environmental aspect</b> <br /><br /><br />
<br />
For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.<br />
<br />
<br /><br /><br />
<br />
<b>Field test</b> <br /><br /><br />
<br />
Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient. <br />
<br />
</div><br />
</div><br />
<div id=bottombox><br />
<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
</div><br />
</div><br />
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</div> <br />
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</div><br />
</body><br />
</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T15:24:59Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Blog Entry</a></li><br />
</ul><br />
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<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<br /><br /><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
<br />
<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">tnt.r1-trz</a> (BBa_K216013) <br /><br />
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<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
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<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
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<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
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This part was previously submitted into the Registry <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J58104">(BBa_J58104)</a>. However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
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<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
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<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
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In our project, a fusion protein (Trz, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">BBa_K216004</a>) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
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It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
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<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a>. The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
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<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
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Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
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The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
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where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
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The aldehyde substrate for the reaction can be produced by enzymes encoded by <i>luxCDE</i> (see below).<br />
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The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
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We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
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<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
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Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
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Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
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<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens<br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216008">(BBa_K216008)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>We worked extremely hard to clone <i>luxAB</i> from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned <i>luxAB</i> from Xenorhabdus luminescens. This was successful- hosts expressing <i>luxAB</i> (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal).<br />
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<img src="http://partsregistry.org/wiki/images/8/80/XL_luxAB.jpg"><br />
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<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
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<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
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O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
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<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
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The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
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<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
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Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
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Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
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NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
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<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
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Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
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<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
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This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
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<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
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<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of <i>tnt.r1</i>, <i>trz</i> and <i>luxCDE</i> genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
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<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PompC <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a> and <i>lacZ'</i> <a href="http://partsregistry.org/Part:BBa_J33202">(BBa_J33202)</a>. Used for Miller’s assay.</td><br />
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<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>lacZ'’</i> <a href="http://partsregistry.org/Part:BBa_J33202">(Bba_J33202)</a>. Used for Miller’s assay.</td><br />
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<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>Renilla luciferase</i> <a href="http://partsregistry.org/Part:BBa_J52008">(BBa_J52008)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_J52008">BBa_J52008</a> from the Registry plates as a back-up. </td><br />
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<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and firefly luciferase <a href="http://partsregistry.org/Part:BBa_I712019">(BBa_I712019)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_I712019">BBa_I712019</a> from the Registry plates as a back-up.</td><br />
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<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> <a href="http://partsregistry.org/Part:BBa_K216002">(BBa_ K216002)</a> and <i>trz</i> <a href="http://partsregistry.org/Part:BBa_K216004">(BBa_ K216004)</a>.</td><br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(nitritenitratesensing)Team:Edinburgh/biology(nitritenitratesensing)2009-10-21T13:54:29Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Nitrite/Nitrate-Sensing Pathway</b></font><br />
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<b> Personal note </b> <br /><br /><br />
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!<br />
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<font color="green" style="float:right">Evangeline</font><br />
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<img src="https://static.igem.org/mediawiki/2009/8/84/NitrateDetection.jpg" style="margin-left:-5px;margin-top:60px;"><br />
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TNT filled landmines often leak when it contact with the soil (Jenkins et al., 2001). The chemicals leaking from landmines include 1,3-DNB, 2,4-DNT, and 2,4,6-TNT (Jenkins et al., 2001). Subsequently, these chemicals are degraded to nitrites by soil bacteria (French et al., 1998). As such, we expect a radial diffusion gradients of nitrites around a focal point (the landmine). The size of this radius would depend on both the soil structure (e.g. water content, grain size etc) and the age of the landmine.<br />
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Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the <i>E. coli</i> yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (BBa_K216005). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, <a href="https://2009.igem.org/Team:Edinburgh/biology(results)">click here</a>.<br />
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It is also noteworthy that the nitrite feeding into the nitrite-detecting pathway comes from two sources. First, nitrite that has been degraded by soil bacteria can enter the cell. Second, our E. coli features a nitroreductase (PETN reductase) that degrades TNT to nitrites within the cell (Figure 2).<br />
<br /><br /><br />
In our system, PyeaR will control the expression of a fusion protein that combines <i>Photobacterium phosphoreum luciferase</i> (Accession #: AY341063, BBa_XXXX, hypothetical) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f7/PyeaRrepression.jpg" width="450"><br />
<center><i><b>Figure 1</b> a) PyeaR repression b) Repression relieved by nitrites. Transcription activated.</i></center><br />
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<img src="https://static.igem.org/mediawiki/2009/3/37/PETNReduces.jpg" width="550" height="350"><br />
<center><i><b>Figure 2</b> PETN reduces TNT to nitrite. Along with nitrite in the soil, it binds to the NsrR repressor to activate gene transcription. LuxAB.GFP is produced and blue-green light is emitted.</i></center><br />
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The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT detection.</a>We attempted to clone the genes encoding for <i>Photobacterium phosphoreum</i> luciferase several times, unfortunately this remained unsuccessful. As such, we decided to clone the luciferase-producing genes from <i>Xenorhabdus luminescens</i> (BBa_K216008). We confirmed that this part is functional. </div><br />
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<br /><br /><br />
Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from <i>Xenorhabdus luminescens</i> (Bba_xxxx). The corresponding genes of <i>P. phosphoreum</i> were not used because they are expressed at very low quantities in <i>E. coli</i>. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in <i>E. coli</i> or condon usage bias (by <i>P. phosphoreum</i>). (Lee <i>et al.</i>, 2000 and Miyamoto et al., 1987).<br />
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After consulting our <a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">modellers</a>, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.<br />
<br /><br /><br />
In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following <a href="#"pictures</a>.<br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T13:08:05Z<p>Evangelineoh: </p>
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
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<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">tnt.r1-trz</a> (BBa_K216013) <br /><br />
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<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J58104">(BBa_J58104)</a>. However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trz, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">BBa_K216004</a>) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a>. The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by <i>luxCDE</i> (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens<br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216008">(BBa_K216008)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>We worked extremely hard to clone <i>luxAB</i> from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned <i>luxAB</i> from Xenorhabdus luminescens. This was successful- hosts expressing <i>luxAB</i> (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of <i>tnt.r1</i>, <i>trz</i> and <i>luxCDE</i> genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PompC <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a> and <i>lacZ'</i> <a href="http://partsregistry.org/Part:BBa_J33202">(BBa_J33202)</a>. Used for Miller’s assay.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>lacZ'’</i> <a href="http://partsregistry.org/Part:BBa_J33202">(Bba_J33202)</a>. Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>Renilla luciferase</i> <a href="http://partsregistry.org/Part:BBa_J52008">(BBa_J52008)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_J52008">BBa_J52008</a> from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and firefly luciferase <a href="http://partsregistry.org/Part:BBa_I712019">(BBa_I712019)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_I712019">BBa_I712019</a> from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> <a href="http://partsregistry.org/Part:BBa_K216002">(BBa_ K216002)</a> and <i>trz</i> <a href="http://partsregistry.org/Part:BBa_K216004">(BBa_ K216004)</a>.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T13:01:06Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<br /><br /><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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<br />
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<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
<br />
<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
</div><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J58104">(BBa_J58104)</a>. However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trz, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">BBa_K216004</a>) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a>. The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PompC <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0082">(BBa_R0082)</a> and lacZ’ <a href="http://partsregistry.org/Part:BBa_J33202">(BBa_J33202)</a>. Used for Miller’s assay.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and lacZ’ <a href="http://partsregistry.org/Part:BBa_J33202">(Bba_J33202)</a>. Used for Miller’s assay.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and <i>Renilla luciferase</i> <a href="http://partsregistry.org/Part:BBa_J52008">(BBa_J52008)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_J52008">BBa_J52008</a> from the Registry plates as a back-up. </td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR <a href="http://partsregistry.org/Part:BBa_K216005">(BBa_K216005)</a> and firefly luciferase <a href="http://partsregistry.org/Part:BBa_I712019">(BBa_I712019)</a>. As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived <a href="http://partsregistry.org/Part:BBa_I712019">BBa_I712019</a> from the Registry plates as a back-up.</td><br />
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<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> <a href="http://partsregistry.org/Part:BBa_K216002">(BBa_ K216002)</a> and <i>trz</i> <a href="http://partsregistry.org/Part:BBa_K216004">(BBa_ K216004)</a>.</td><br />
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</tr><br />
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</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(results)Team:Edinburgh/biology(results)2009-10-21T12:39:57Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Results</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<b> Personal Note </b> <br /><br /><br />
One of the best things about iGEM is that you get to learn <b>a lot</b> from your peers. Biologists learn about modelling, engineers learn about biology...and we all have to learn a little <i>HTML</i>. The learning can be intensely stressful at times but also very satisfying.<br />
<br /><br /><br />
<font color="green" style="float:right">Evangeline</font><br />
<br />
<br /><br /><br />
What is even more important, we learn from each other not just the technical knowledge, but something what is more precious for me - life views. Getting other people's life views you can extend and adjust your personal life view, make it bigger, smarter and more adaptive.<br />
<br /><br /><br />
<font color="green" style="float:right">JD</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/7/71/OmpcCharachterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/1/13/Ompc-promoter.jpg"> </center><br />
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<br />
We used Jason Kelly's promoter characterization kit to characterize the <i>ompC</i> promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.<br />
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<img src="https://static.igem.org/mediawiki/2009/b/bb/YeaRPRomoterCharacterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<br /><br />
<b> Miller Test </b> <br /><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/e/ef/Millertest.jpg"> </center> <br /><br /><br />
<br />
The classical Miller's Assay was used to characterize the <i>yeaR</i> promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.<br />
<br />
<br /><br /><br />
<br />
<b>PyeaR characterization with fluorescence using Jason Kelly's kit</b> <br /><br />
<br><br />
<center> <img src="http://partsregistry.org/wiki/images/a/a9/Pyear_characterization.jpg"> </center> <br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8d/EdinburghFurtherWork.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed! <br />
<br />
<br /><br /><br />
<br />
<b>Testing TNT receptors</b><br /><br /><br />
<br />
First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.<br />
<br />
<br /><br /><br />
<br />
<b>Environmental aspect</b> <br /><br /><br />
<br />
For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.<br />
<br />
<br /><br /><br />
<br />
<b>Field test</b> <br /><br /><br />
<br />
Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient. <br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T09:34:52Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
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<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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</tr><br />
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<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T09:31:33Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<br /><br /><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
<br />
<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
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<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
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<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
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<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
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</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T09:30:27Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
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<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
<br />
</td><br />
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<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-21T09:30:01Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Blog Entry</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
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<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - functional and/or characterized BioBrick<br /><br />
</div><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></br><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Supervisors</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/8/85/EdinburghChris_french.png" width="150" height="200" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Chris French <br /><br />
<b> &nbsp;&nbsp; Department:</b> Biology <br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>C.French<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">ed.ac.uk<br /><br /><br />
&nbsp;&nbsp;&nbsp;My first degree is in Bioprocess Engineering, and my Ph.D. is in Microbial Biotechnology<br />
(specifically, &nbsp;&nbsp;&nbsp;biotransformations of natural products for the chemical industry). I now<br />
lecture in general and applied &nbsp;&nbsp;&nbsp;microbiology &nbsp;&nbsp;&nbsp;and biotechnology in the School of Biological<br />
Sciences, University of Edinburgh. My research is &nbsp;&nbsp;&nbsp;mainly concerned with the development<br />
of microbial systems for industrial and environmental applications. I &nbsp;&nbsp;&nbsp;have been<br />
supervising Edinburgh's IGEM entries since 2006. I think BioBricks are a great tool for<br />
the &nbsp;&nbsp;&nbsp;development of new biological systems; my Ph.D. students are applying them to the<br />
development of biosensors &nbsp;&nbsp;&nbsp;and systems for conversion of lignocellulosic biomass to useful<br />
products.<br /><br />
<br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/7/7f/Alistair_elfick.jpg" width="150" height="150" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Alistair Elfick <br /><br />
<b> &nbsp;&nbsp; Department:</b> Engineering <br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>Alistair.Elfick<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">ed.ac.uk<br /><br /><br /><br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/9/9e/Vincent_danos.jpg" width="150" height="150" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Vincent Danos <br /><br />
<b> &nbsp;&nbsp; Department:</b> Informatics <br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>vincent.danos<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br /><br /><br />
&nbsp;&nbsp;&nbsp;Vincent is a prof of computational systems biology with special interests in the architecture of natural/synthetic &nbsp;&nbsp;&nbsp;protein networks.<br /><br /><br />
<br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29"> Team Members </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Advisors</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/8/8e/EdinburghKim_de_mora.png" width="150" height="187" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Kim de Mora <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hi! I'm currently a PhD candidate at the University of Edinburgh. My research interests include engineering &nbsp;&nbsp;&nbsp;peroxisomes in <i>S. cerevisiae</i> and investigating standardization questions in synthetic biology. I was a member of &nbsp;&nbsp;&nbsp;the 2006 Edinburgh iGEM team when we designed and built the arsenic biosensor. I've since been an advisor for &nbsp;&nbsp;&nbsp;the 2007, 2008 and 2009 teams.<br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>kimdemora<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br />
<br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/5b/EdinburghLucas_black.jpg" width="150" height="98" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Lucas Black<br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>s.black<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">ed.ac.uk<br />
<br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/0/0a/Calvert_picture.jpg" width="150" height="180" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Jane Calvert <br /><br /><br />
&nbsp;&nbsp;&nbsp;Jane is a sociologist of science, interested in<br />
all things to do with synthetic biology<br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>jane.calvert<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">ed.ac.uk<br />
<br /><br /><br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/6d/EF_photo.jpg" width="150" height="180" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Emma Frow <br /><br /><br />
&nbsp;&nbsp;&nbsp;Emma is Jane's sidekick. <br /><br /><br />
&nbsp;&nbsp;&nbsp;After years at the lab bench, Emma has recently<br />
ventured into the sociology of science. Her research interests are &nbsp;&nbsp;&nbsp;in synthetic biology and plant biotechnology.<br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>emma.frow<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">ed.ac.uk<br />
<br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/1/14/Damian_barnard.JPG" width="150" height="165" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> DK Barnard <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hi,I'm Damian. I am a PhD student working on a synthetic biology approach to cellulose<br />
degradation. Being &nbsp;&nbsp;&nbsp;involved with iGEM for the first time as an advisor was great fun.<br />
Over the past few weeks I've been helping out the &nbsp;&nbsp;&nbsp;team around the lab and offering what<br />
good (or bad) advice I could. The team worked extremely well in the lab &nbsp;&nbsp;&nbsp;and very quickly<br />
got the hang of BioBricking. Best of luck at the prize giving guys. Enjoy the rest of the<br />
tour around &nbsp;&nbsp;&nbsp;the wiki!!<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: dkbarnard</b><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">googlemail.com<br /><br /><br /><br /><br />
<br />
</div><br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Blog Entry</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<br />
<div style="width:100%"><br />
<br />
<br /><br /><br /><br />
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<div id="text" style="margin-left:5px;margin-right:5px;"><br />
<font color="#ffffff">Our fancy notebook details work in the weblab. <b>Scroll using the arrows for June-August entries.</b> Days which have entries are highlighted in a different colour.</font><br />
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<div id="Edinburghfooter"><center><font color="#ffffff">iGem Team 2009 Edinburgh</font></center></div><br />
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</html></div>Evangelineohhttp://2009.igem.org/File:Pompc_characterization.jpgFile:Pompc characterization.jpg2009-10-20T15:35:36Z<p>Evangelineoh: </p>
<hr />
<div></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-20T14:08:46Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
<br />
<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
<br />
<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-20T14:05:15Z<p>Evangelineoh: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<br /><br /><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
<br />
<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
<br />
</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
<br />
</div><br />
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<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
<br />
<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="tntr1trz">tnt.r1-trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013"</a>(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
<div id=bottombox><br />
<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
</div><br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-20T13:58:29Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<br /><br /><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the links below the images of each part</b></center><br /><br /><br />
<br />
<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
<br />
</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216015">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="tntr1trz">TNT.R1-Trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216013</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
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</tr><br />
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</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-20T13:56:14Z<p>Evangelineoh: </p>
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<div id=mainmenu><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28summary%29">DEMOCS Card Game</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<br /><br /><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<center><b><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Edinburgh">Click here</a> to view our parts on the Registry or click on the link below the images of each part</b></center><br /><br /><br />
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<div id="left" style="width:40%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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</div><br />
<div id="right" style="width:59%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
<br />
<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein<br> </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
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</td><br />
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<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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</font><br />
</td><br />
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<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i><br></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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</tr><br />
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<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:(BBa_K216015)">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="tntr1trz">TNT.R1-Trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:(BBa_K216013)">(BBa_K216013)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-20T13:49:10Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<center><b>See Our Parts on the Registry of Standard Biological Parts by clicking Biobricks' Codes</b></center><br /><br /><br />
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<b>Quick links:</b> <br /><br /><br />
<a href="#tnt">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#luxabxeno">luxAB from Xenorhabdus luminescens</a> (BBa_K216008)<br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pyearrenilla">PyeaR-Renilla luciferase</a> (BBa_K216014) <br /><br />
<a href="#pyearfirefly">PyeaR-firefly luciferase</a> (BBa_K216015) <br /><br />
<a href="#tntr1trz">TNT.R1-Trz</a> (BBa_K216013) <br /><br />
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<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
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<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
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This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
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<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
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<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
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In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
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It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
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<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
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<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from Enterobacter cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
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Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
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The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
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where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
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The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
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The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
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We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
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<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
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Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
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Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
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<td><center><b><a name="luxabxeno">luxAB from Xenorhabdus luminescens </a> (BBa_K216008)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>We worked extremely hard to clone luxAB from Photobacterium phosphoreum, unfortunately, we were unsuccessful. As such, we cloned luxAB from Xenorhabdus luminescens. This was successful- hosts expressing luxAB (from X, luminescens) under the control of the yeaR promoter emitted light in the presence of nitrites and aldehyde substrate (decanal). <br />
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<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
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<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
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O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
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<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
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The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
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<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
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Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
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Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
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<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
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<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
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This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
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<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
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<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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<td><center><b><a name="pyearrenilla">PyeaR-<i>Renilla luciferase</i> </a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216014">(BBa_K216014)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and <i>Renilla luciferase</i> (BBa_J52008). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_J52008 from the Registry plates as a back-up. </td><br />
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<br />
<tr><br />
<td><center><b><a name="pyearfirefly">PyeaR-<i>firefly luciferase</i></a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:(BBa_K216015)">(BBa_K216015)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and firefly luciferase (BBa_I712019). As we were enountered great difficulty in cloing <i>Photobacterium phosphoreum luciferase</i>, we revived BBa_I712019 from the Registry plates as a back-up.</td><br />
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<tr><br />
<td><center><b><a name="tntr1trz">TNT.R1-Trz</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:(BBa_K216013)">(BBa_K216013)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying <i>tnt.r1</i> (BBa_ K216002) and <i>trz</i> (BBa_ K216004).</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(solvedproblems)Team:Edinburgh/biology(solvedproblems)2009-10-20T11:40:58Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Problem Solving and Tips</b></font><br />
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<div id=Edinburghcontent><br />
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<b> Personal note </b> <br /><br /><br />
"There is a time for everything...a time to tear down and a time to build..." - Ecclesiastes 3<br />
<br><br><br />
Synthetic biology is all about construction and deconstruction. It is about tearing down and building up, both at the molecular level and level of generating useful applications. Zoom in, zoom out, step forward, step backward...enjoy.<br />
<br /><br /><br />
<font color="green" style="float:right">Evangeline</font><br />
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<img src="https://static.igem.org/mediawiki/2009/d/df/ProblemsAndSolutions.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
With the revolutionary invention of biobricking techniques using the EcoRI, XbaI, SpeI and PstI restriction sites, constructing biobrick parts seems straight-forward enough. Yet, all self-respecting scientists recognize that things are never as simple as following a protocol. Here, we compile as list of problems we encountered during our project, solutions we found or suggested solutions and tips that will make your future biobricking experiences even fuller.<br />
<br /><br /><br />
<b>Low cell transformation efficiency. Repeated miniprep and sequencing did not identify correct construct.</b> <br /><br /> If you are encountering this problem and have used the classical method involving double digestion of your vector and desired insert followed by ligation then transformation, consider fusion PCR! We used this method and found that the Biobricking efficiency is significantly higher. A word of caution, do ensure that your competent cells are <i>truly</i> competent before proceeding. As our very wise supervisor once advised, “if something goes wrong, the first thing you do is to assume that you mucked up”.<br />
<br /><br /><br />
<a href="https://static.igem.org/mediawiki/2009/2/27/FusionPCR.jpg"><img src="https://static.igem.org/mediawiki/2009/2/27/FusionPCR.jpg" width="775" border="0"></a><br />
<br />
<br /><br /><br />
<b>Strategic biobricking to improve efficiency</b><br />
<br /><br /><br />
If you are making a composite biobrick part from parts A and B, and A causes transformants to confer an easily detectable phenotype such as fluorescence or blue colouration, consider inserting biobrick part A into a vector already carrying B rather than vice versa. In this way, you know immediately if your ligation has been successful. This can potentially save you from minipreping and sequencing erroneous constructs.<br />
<br /><br /><br />
Another issue that is seemingly obvious but might be overlooked when constructing composite biobrick parts is trying to insert a biobrick promoter upstream of a coding sequence carried on a vector. Promoter sequences are often short (<150 bp) and any size changes caused by successful insertion of the promoter in the vector will be difficult to detect on an agarose gel. For efficient screening, insert the coding sequence downstream of the promoter instead. <br />
<br />
<br /><br /><br />
<b>No PCR product when cell extract was used as template</b><br />
<br /><br /><br />
In our project, we tried cloning the luciferase genes (<i>luxAB</i>) from <i>Photobacterium phosphoreum</i>. After countless (trust us) attempts, Vas (devoted Edinburgh iGEMer) decided to use <i>P. phosphoreum</i> cell extract that had been left out on the bench for 3 days as the template. Violà! We eventually got a PCR product. Instead of waiting 3 days for your cells to die, we suggest boiling them for 10 minutes before running the PCR.<br />
<br /><br /><br />
Tricky PCR might also be solved by experimenting with lower annealing temperature or high concentration of magnesium sulphate. The reason being, biobrick compliant primers carry the prefix and suffix, as such, primer specificity to DNA sequence is lowered. Adjusting PCR conditions will improve primer annealing.<br />
<br />
<br /><br /><br />
<b>Primer Design</b><br />
<br /><br /><br />
Did you know, there’s a web-based software that generates reverse complement sequences. Find it here: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a>. Silly, but we didn’t know. <br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/team(teammembers)Team:Edinburgh/team(teammembers)2009-10-19T15:42:36Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Team Members</b></font><br />
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Here we are! Edinburgh University 2009 iGEM Team :P This our our video team introduction. Every single person who had impact on our team project has his own small video snippet. Check this out!<br /><br />
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<img src="https://static.igem.org/mediawiki/2009/4/42/Vas.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Vasilis Andreou <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hey guys my name is Vasilis Andreou. Something you don't know about me is that my house in Nicosia, Cyprus is about 2 &nbsp;&nbsp;&nbsp;minutes from the borders dividing the town, and in the opposite direction is about 2 minutes from the country's prison. See you &nbsp;&nbsp;&nbsp;in MIT!!<br /><br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>V.Andreou<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Evangeline JiaLing Oh <br /><br /><br />
&nbsp;&nbsp;&nbsp;I was born and bred in Singapore. Apart from ice-cream, I love gene regulatory networks. Sometime in the near future, I hope &nbsp;&nbsp;&nbsp;to be BioBricking in eukaryotic cells. Have fun and break a leg in MIT.<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>E.J.Oh-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/b/b8/EdinburghDasha%28edited%29.jpg" width="150" height="150" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Dasha <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hi I am Dasha and I love biology. iGEM has been the most exhilarating and challenging<br />
project I have ever done, and it made me &nbsp;&nbsp;&nbsp;realize how many possibilities are presented to<br />
me by what I study! <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>D.Ausiannikava<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c5/Juliaedinburgh.jpg" width="150" height="280" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Julia <br /><br /><br />
&nbsp;&nbsp;&nbsp;My name is Julia and I was born in Kiev, Ukraine. I moved to Belgium at a young age and since then I have had the pleasure of &nbsp;&nbsp;&nbsp;being a citizen of the world, having lived in Ireland, UK and the Middle East—and the list keeps growing! I have degrees in &nbsp;&nbsp;&nbsp;Biotechnology and Drug Discovery, and particularly love everything gene-related. <br /><br /><br />
&nbsp;&nbsp;&nbsp;To me, IGEM conveys an emotion. A mixture of excitement, uncertainty and, most intriguingly, empowerment. If you, as a &nbsp;&nbsp;&nbsp;biologist, ever doubt the practical potential of your knowledge, I challenge you to take part in this competition—you will &nbsp;&nbsp;&nbsp;instantly understand what kind of powerful sci-fi stuff lurks within your brain! <br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>J.Skripka<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<b> &nbsp;&nbsp; Name: </b> Winston Ho <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello there! Names Winston, born in the 'deen, which is up in the North-East of Scotland. I'm studying chemical engineering, &nbsp;&nbsp;&nbsp;having just started my 4th year at Edinburgh. Can't wait to join the oil industry ;) I also have a soft spot for biology =) CU at MIT!<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b> W.Ho-3<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f5/Rachel.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rachel "Lady GaGa" Morrison <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello, my name is Rachel though over the course of the summer my lovely team mates have called me a variety of things &nbsp;&nbsp;&nbsp;including Lady Gaga. I am currently in my final year of a master’s of Chemical Engineering though I have spent most of the &nbsp;&nbsp;&nbsp;summer learning how to model gene expression networks, program in JAVA and MATLAB and have also picked up some &nbsp;&nbsp;&nbsp;biology along the way! <br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>R.A.Morrison-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/d/d5/TeamInformaticians.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<b> &nbsp;&nbsp; Name: </b> JD <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email:</b> jan.domozilov<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rohin <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8b/TeamGueststars.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<b> &nbsp;&nbsp; Name: </b> Sam Kammerling<br />
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&nbsp;&nbsp;&nbsp;I'm Sam, and I have been singing theme tunes for scientists for as long as I can <br />
remember. Growing up in the English &nbsp;&nbsp;&nbsp;countryside it was every child's dream to have your <br />
voice heard on the international scientific stage, and now this dream has &nbsp;&nbsp;&nbsp;come true <br />
thanks to the Edinburgh iGem team. Singing about bacteria is my passion, and I hope I <br />
inspire others to be as &nbsp;&nbsp;&nbsp;passionate about bacteria as me. Big love peeps x<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email:</b> samkam86<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">hotmail.com<br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c3/KacperEdinburgh.jpg" width="150" height="227" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Kacper Rogala <br /><br /><br />
&nbsp;&nbsp;&nbsp;I study Biochemistry, love film and good food! I was engaged to film, act in and produce the team's awesome promotional &nbsp;&nbsp;&nbsp;video. This picture of me was taken during the "explosion scene". I am also a hairy chewbacca.<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email:</b> skurwiec<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br />
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<img src="https://static.igem.org/mediawiki/2009/d/de/Teamgeogaphy.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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We are very international team, so we thought it might be interesting for you to know bit more about places we all came from. Our team consists of 8 undergraduates from 7 different countries. <br /><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/team(teammembers)Team:Edinburgh/team(teammembers)2009-10-19T15:33:56Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29"> Team Members </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29"> Advisors </a> </div><br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28edinburghuniversity%29"> Edinburgh University </a> </div> <br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29"> Acknowledgements </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Team Members</b></font><br />
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<div id=Edinburghcontent><br />
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Here we are! Edinburgh University 2009 iGEM Team :P This our our video team introduction. Every single person who had impact on our team project has his own small video snippet. Check this out!<br /><br />
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<img src="https://static.igem.org/mediawiki/2009/f/fa/TeamBiologists.jpg" style="margin-left:-5px;margin-top:10px;"><br />
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<img src="https://static.igem.org/mediawiki/2009/4/42/Vas.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Vasilis Andreou <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hey guys my name is Vasilis Andreou. Something you don't know about me is that my house in Nicosia, Cyprus is about 2 &nbsp;&nbsp;&nbsp;minutes from the borders dividing the town, and in the opposite direction is about 2 minutes from the country's prison. See you &nbsp;&nbsp;&nbsp;in MIT!!<br /><br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>V.Andreou<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Evangeline JiaLing Oh <br /><br /><br />
&nbsp;&nbsp;&nbsp;I was born and bred in Singapore. Apart from ice-cream, I love gene regulatory networks. Sometime in the near future, I hope &nbsp;&nbsp;&nbsp;to be BioBricking in eukaryotic cells. Have fun and break a leg in MIT.<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>E.J.Oh-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/b/b8/EdinburghDasha%28edited%29.jpg" width="150" height="150" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Dasha <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hi I am Dasha and I love biology. iGEM has been the most exhilarating and challenging<br />
project I have ever done, and it made me &nbsp;&nbsp;&nbsp;realize how many possibilities are presented to<br />
me by what I study! <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>D.Ausiannikava<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c5/Juliaedinburgh.jpg" width="150" height="280" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Julia <br /><br /><br />
&nbsp;&nbsp;&nbsp;My name is Julia and I was born in Kiev, Ukraine. I moved to Belgium at a young age and since then I have had the pleasure of &nbsp;&nbsp;&nbsp;being a citizen of the world, having lived in Ireland, UK and the Middle East—and the list keeps growing! I have degrees in &nbsp;&nbsp;&nbsp;Biotechnology and Drug Discovery, and particularly love everything gene-related. <br /><br /><br />
&nbsp;&nbsp;&nbsp;To me, IGEM conveys an emotion. A mixture of excitement, uncertainty and, most intriguingly, empowerment. If you, as a &nbsp;&nbsp;&nbsp;biologist, ever doubt the practical potential of your knowledge, I challenge you to take part in this competition—you will &nbsp;&nbsp;&nbsp;instantly understand what kind of powerful sci-fi stuff lurks within your brain! <br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>J.Skripka<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<div id="text" style="margin-left:20px;margin-top:10px;height:530px;"><br />
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<img src="https://static.igem.org/mediawiki/2009/d/db/WinyEdinburgh.jpg" width="150" height="281" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Winston Ho <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello there! Names Winston, born in the 'deen, which is up in the North-East of Scotland. I'm studying chemical engineering, &nbsp;&nbsp;&nbsp;having just started my 4th year at Edinburgh. Can't wait to join the oil industry ;) I also have a soft spot for biology =) CU at MIT!<br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b> W.Ho-3<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f5/Rachel.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rachel "Lady GaGa" Morrison <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello, my name is Rachel though over the course of the summer my lovely team mates have called me a variety of things &nbsp;&nbsp;&nbsp;including Lady Gaga. I am currently in my final year of a master’s of Chemical Engineering though I have spent most of the &nbsp;&nbsp;&nbsp;summer learning how to model gene expression networks, program in JAVA and MATLAB and have also picked up some &nbsp;&nbsp;&nbsp;biology along the way! <br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>R.A.Morrison-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/d/d5/TeamInformaticians.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;height:480px;"><br />
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<img src="https://static.igem.org/mediawiki/2009/4/42/Vas.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> JD <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email:</b> jan.domozilov<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rohin <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8b/TeamGueststars.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;height:410px;"><br />
<img src="https://static.igem.org/mediawiki/2009/c/cf/Sam_Kammerling.jpg" width="150" height="200" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Sam Kammerling<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;I'm Sam, and I have been singing theme tunes for scientists for as long as I can <br />
remember. Growing up in the English &nbsp;&nbsp;&nbsp;countryside it was every child's dream to have your <br />
voice heard on the international scientific stage, and now this dream has &nbsp;&nbsp;&nbsp;come true <br />
thanks to the Edinburgh iGem team. Singing about bacteria is my passion, and I hope I <br />
inspire others to be as &nbsp;&nbsp;&nbsp;passionate about bacteria as me. Big love peeps x<br />
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<br /><br />
<br /><br />
<br />
<b> &nbsp;&nbsp;&nbsp;Contact email:</b> samkam86<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">hotmail.com<br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c3/KacperEdinburgh.jpg" width="150" height="227" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Kacper Rogala <br /><br /><br />
&nbsp;&nbsp;&nbsp;I study Biochemistry, love film and good food! I was engaged to film, act in and produce the team's awesome promotional &nbsp;&nbsp;&nbsp;video. This picture of me was taken during the "explosion scene". I am also a hairy chewbacca.<br />
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<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/d/de/Teamgeogaphy.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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We are very international team, so we thought it might be interesting for you to know bit more about places we all came from. Our team consists of 8 undergraduates from 7 different countries. <br /><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:53:53Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
</div><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a><br><a href="http://partsregistry.org/Part:BBa_J23105">(BBa_J23105)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216012">(BBa_K216012)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216011">(BBa_K216011)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/54/Edinburgheyfpresult.jpg"></center></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216010">(BBa_K216010)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a><br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216009">(BBa_K216009)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:49:36Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
</div><br />
<br />
<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
<br />
</div><br />
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a><br> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216007">(BBa_K216007)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:48:15Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005">(BBa_K216005)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
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Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:46:29Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
</div><br />
<br />
<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
<br />
</div><br />
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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<td><center><b><a name="onr">onr</a> <br><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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</tr><br />
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</table><br />
<br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:45:16Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216006">(BBa_K216006)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
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<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:43:34Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
</div><br />
<br />
<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
<br />
</div><br />
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> <a href="http://partsregistry.org/Part:BBa_E0430">(BBa_E0430)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:40:32Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216003">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><br><a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
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<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
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<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
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Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
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The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
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<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:37:40Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
</div><br />
<br />
<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
</div><br />
<br />
</div><br />
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216003</a>)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216004">(BBa_K216004)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a><a href="<a href="http://partsregistry.org/Part:BBa_R0082">(BBa_R0082)</a></b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T14:33:49Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K216002">BBa_K216002</a> & BBa_K216003)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> (BBa_K216004)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a> (BBa_R0082)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
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<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
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<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Acknowledgements</b></font><br />
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<div id=Edinburghcontent><br />
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<br />
We would also like to thank a lot of people, without their contributiosn we would not have been able to proceed as much as we have done:<br />
<p><br />
<b>Dr Homme W. Hellinga</b> from Duke University for providing us with <i>tnt.r1</i> and <i>tnt.r3</i> in their original plasmids.<br />
<p><br />
<b>Dr Gerald Hazelbauer</b> from the University of University of Missouri-Columbia, and his assistant Lilly Angela for providing us with the construct of the fusion protein TrZ.<br />
<p><br />
<b>Dr H.J.E Beaumont</b> from the Institute of Biology, Leiden for providing us with <i>nir</i> promoter and <i>nsrR</i> construct from <i>N. europea</i><br />
<p><br />
<b>Dr Donald Bruce</b> from Edinethics for introducing us to the Demos card game, and allowing us to adapt his basic prototypes for our wiki.<br />
<p><br />
<b>Ms Elaine Murphy</b> from the Centre for Intelligent Systems and their Applications for giving us a tutorial on how to use Cellucidate.<br />
<p><br />
<b>Mr Ty Thomson</b> for attending to numerous questions considering the Cellucidate platform. <br />
<p><br />
<b>Ms Sahreena Lakhudi and Mr Chao-Kuo Liu</b> for putting up with our questions (intelligent/ stupid) in the lab all summer.<br />
<p><br />
And of course all the ladies working in the media prep room on the 8th floor of Darwin Building.<br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(results)Team:Edinburgh/biology(results)2009-10-19T13:38:07Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Results</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<b> Personal Note </b> <br /><br /><br />
One of the best things about iGEM is that you get to learn <b>a lot</b> from your peers. Biologists learn about modelling, engineers learn about biology...and we all have to learn a little <i>HTML</i>. The learning can be intensely stressful at times but also very satisfying.<br />
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<font color="green" style="float:right">Evangeline</font><br />
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<img src="https://static.igem.org/mediawiki/2009/7/71/OmpcCharachterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/1/13/Ompc-promoter.jpg"> </center><br />
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We used Jason Kelly's promoter characterization kit to characterize the <i>ompC</i> promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.<br />
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<img src="https://static.igem.org/mediawiki/2009/b/bb/YeaRPRomoterCharacterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<b> Miller Test </b> <br /><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/e/ef/Millertest.jpg"> </center> <br /><br /><br />
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The classical Miller's Assay was used to characterize the <i>yeaR</i> promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.<br />
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<b> Jason Kelly Assay </b> <br /><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/d/d8/Jasonkelly.jpg"> </center> <br /><br /><br />
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From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.<br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8d/EdinburghFurtherWork.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed! <br />
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<b>Testing TNT receptors</b><br /><br /><br />
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First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.<br />
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<b>Environmental aspect</b> <br /><br /><br />
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For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.<br />
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<b>Field test</b> <br /><br /><br />
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Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient. <br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(nitritenitratesensing)Team:Edinburgh/biology(nitritenitratesensing)2009-10-19T13:28:31Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Nitrite/Nitrate-Sensing Pathway</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!<br />
<br /><br /><br />
<font color="green" style="float:right">Evangeline</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/8/84/NitrateDetection.jpg" style="margin-left:-5px;margin-top:60px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:45px;"><br />
<br />
TNT filled landmines often leak when it contact with the soil (Jenkins et al., 2001). The chemicals leaking from landmines include 1,3-DNB, 2,4-DNT, and 2,4,6-TNT (Jenkins et al., 2001). Subsequently, these chemicals are degraded to nitrites by soil bacteria (French et al., 1998). As such, we expect a radial diffusion gradients of nitrites around a focal point (the landmine). The size of this radius would depend on both the soil structure (e.g. water content, grain size etc) and the age of the landmine.<br />
<br /><br /><br />
Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the <i>E. coli</i> yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (BBa_K216005). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, <a href="https://2009.igem.org/Team:Edinburgh/biology(results)">click here</a>.<br />
<br /><br /><br />
<br />
<div id="left" style="width:42%;padding-bottom:45px;height:160px;"><br />
It is also noteworthy that the nitrite feeding into the nitrite-detecting pathway comes from two sources. First, nitrite that has been degraded by soil bacteria can enter the cell. Second, our E. coli features a nitroreductase (PETN reductase) that degrades TNT to nitrites within the cell (Figure 2).<br />
<br /><br /><br />
In our system, PyeaR will control the expression of a fusion protein that combines <i>Photobacterium phosphoreum luciferase</i> (Accession #: AY341063, BBa_XXXX, hypothetical) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.<br />
<br />
</div><br />
<br />
<div id="right" style="width:57%;padding-bottom:45px;height:160px;"><br />
<img src="https://static.igem.org/mediawiki/2009/f/f7/PyeaRrepression.jpg" width="450"><br />
<center><i><b>Figure 1</b> a) PyeaR repression b) Repression relieved by nitrites. Transcription activated.</i></center><br />
</div><br />
<br />
<div id="left" style="width:69%;padding-bottom:25px;margin-top:5px;height:410px;"><br />
<img src="https://static.igem.org/mediawiki/2009/3/37/PETNReduces.jpg" width="550" height="350"><br />
<center><i><b>Figure 2</b> PETN reduces TNT to nitrite. Along with nitrite in the soil, it binds to the NsrR repressor to activate gene transcription. LuxAB.GFP is produced and blue-green light is emitted.</i></center><br />
</div><br />
<br />
<div id="right" style="width:30%;padding-bottom:25px;margin-top:5px;height:410px;"><br />
<br><br><br />
The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT detection.</a>We attempted to clone the genes encoding for <i>Photobacterium phosphoreum</i> luciferase several times, unfortunately this remained unsuccessful. As such, we decided to clone the luciferase-producing genes from <i>Xenorhabdus luminescens</i> (BBa_K216008). We confirmed that this part is functional. </div><br />
<br />
<br /><br /><br />
Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from <i>Xenorhabdus luminescens</i> (Bba_xxxx). The corresponding genes of <i>P. phosphoreum</i> were not used because they are expressed at very low quantities in <i>E. coli</i>. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in <i>E. coli</i> or condon usage bias (by <i>P. phosphoreum</i>). (Lee <i>et al.</i>, 2000 and Miyamoto et al., 1987).<br />
</div><br />
<br />
<br /><br /><br />
After consulting our <a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">modellers</a>, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.<br />
<br /><br /><br />
In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following <a href="#"pictures</a>.<br />
<br />
</div><br />
<br />
</div><br />
<div id=bottombox><br />
<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
</div><br />
</div><br />
<br />
</div> <br />
<br />
</div><br />
</body><br />
</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(nitritenitratesensing)Team:Edinburgh/biology(nitritenitratesensing)2009-10-19T13:22:02Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Nitrite/Nitrate-Sensing Pathway</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!<br />
<br /><br /><br />
<font color="green" style="float:right">Evangeline</font><br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/8/84/NitrateDetection.jpg" style="margin-left:-5px;margin-top:60px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:45px;"><br />
<br />
TNT filled landmines often leak when it contact with the soil (Jenkins et al., 2001). The chemicals leaking from landmines include 1,3-DNB, 2,4-DNT, and 2,4,6-TNT (Jenkins et al., 2001). Subsequently, these chemicals are degraded to nitrites by soil bacteria (French et al., 1998). As such, we expect a radial diffusion gradients of nitrites around a focal point (the landmine). The size of this radius would depend on both the soil structure (e.g. water content, grain size etc) and the age of the landmine.<br />
<br /><br /><br />
Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the <i>E. coli</i> yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (BBa_K216005). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, <a href="https://2009.igem.org/Team:Edinburgh/biology(results)">click here</a>.<br />
<br /><br /><br />
<br />
<div id="left" style="width:42%;padding-bottom:45px;height:160px;"><br />
It is also noteworthy that the nitrite feeding into the nitrite-detecting pathway comes from two sources. First, nitrite that has been degraded by soil bacteria can enter the cell. Second, our E. coli features a nitroreductase (PETN reductase) that degrades TNT to nitrites within the cell (Figure 2).<br />
<br /><br /><br />
In our system, PyeaR will control the expression of a fusion protein that combines <i>Photobacterium phosphoreum luciferase</i> (Accession #: AY341063, BBa_XXXX, hypothetical) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.<br />
<br />
</div><br />
<br />
<div id="right" style="width:57%;padding-bottom:45px;height:160px;"><br />
<img src="https://static.igem.org/mediawiki/2009/f/f7/PyeaRrepression.jpg" width="450"><br />
<center><i><b>Figure 1</b> a) PyeaR repression b) Repression relieved by nitrites. Transcription activated.</i></center><br />
</div><br />
<br />
<div id="left" style="width:69%;padding-bottom:25px;margin-top:5px;height:410px;"><br />
<img src="https://static.igem.org/mediawiki/2009/3/37/PETNReduces.jpg" width="550" height="350"><br />
<center><i><b>Figure 2</b> PETN reduces TNT to nitrite. Along with nitrite in the soil, it binds to the NsrR repressor to activate gene transcription. LuxAB.GFP is produced and blue-green light is emitted.</i></center><br />
</div><br />
<br />
<div id="right" style="width:30%;padding-bottom:25px;margin-top:5px;height:410px;"><br />
The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT detection</a>. We attempted to clone the genes encoding for <i>Photobacterium phosphoreum<i> luciferase several times, unfortunately this remained unsuccessful. As such, we decided to clone the luciferase-producing genes from <i>Xenorhabdus luminescens</i> (BBa_K216008). We confirmed that this part is functional. <br />
<br /><br /><br />
Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from <i>Xenorhabdus luminescens</i> (Bba_xxxx). The corresponding genes of <i>P. phosphoreum</i> were not used because they are expressed at very low quantities in <i>E. coli</i>. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in <i>E. coli</i> or condon usage bias (by <i>P. phosphoreum</i>). (Lee <i>et al.</i>, 2000 and Miyamoto et al., 1987).<br />
</div><br />
<br />
<br /><br /><br />
After consulting our <a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">modellers</a>, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.<br />
<br /><br /><br />
In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following <a href="#"pictures</a>.<br />
<br />
</div><br />
<br />
</div><br />
<div id=bottombox><br />
<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
</div><br />
</div><br />
<br />
</div> <br />
<br />
</div><br />
</body><br />
</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(nitritenitratesensing)Team:Edinburgh/biology(nitritenitratesensing)2009-10-19T13:12:57Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Nitrite/Nitrate-Sensing Pathway</b></font><br />
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<b> Personal note </b> <br /><br /><br />
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!<br />
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<font color="green" style="float:right">Evangeline</font><br />
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TNT filled landmines often leak when it contact with the soil (Jenkins et al., 2001). The chemicals leaking from landmines include 1,3-DNB, 2,4-DNT, and 2,4,6-TNT (Jenkins et al., 2001). Subsequently, these chemicals are degraded to nitrites by soil bacteria (French et al., 1998). As such, we expect a radial diffusion gradients of nitrites around a focal point (the landmine). The size of this radius would depend on both the soil structure (e.g. water content, grain size etc) and the age of the landmine.<br />
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Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the <i>E. coli</i> yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (BBa_K216005). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, <a href="https://2009.igem.org/Team:Edinburgh/biology(results)">click here</a>.<br />
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It is also noteworthy that the nitrite feeding into the nitrite-detecting pathway comes from two sources. First, nitrite that has been degraded by soil bacteria can enter the cell. Second, our E. coli features a nitroreductase (PETN reductase) that degrades TNT to nitrites within the cell (Figure 2).<br />
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In our system, PyeaR will control the expression of a fusion protein that combines <i>Photobacterium phosphoreum luciferase</i> (Accession #: AY341063, BBa_XXXX, hypothetical) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.<br />
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<center><i><b>Figure 1</b> a) PyeaR repression b) Repression relieved by nitrites. Transcription activated.</i></center><br />
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<img src="https://static.igem.org/mediawiki/2009/3/37/PETNReduces.jpg" width="550" height="350"><br />
<center><i><b>Figure 2</b> PETN reduces TNT to nitrite. Along with nitrite in the soil, it binds to the NsrR repressor to activate gene transcription. LuxAB.GFP is produced and blue-green light is emitted.</i></center><br />
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The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT detection</a>. <br />
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Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from <i>Xenorhabdus luminescens</i> (Bba_xxxx). The corresponding genes of <i>P. phosphoreum</i> were not used because they are expressed at very low quantities in <i>E. coli</i>. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in <i>E. coli</i> or condon usage bias (by <i>P. phosphoreum</i>). (Lee <i>et al.</i>, 2000 and Miyamoto et al., 1987).<br />
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After consulting our <a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">modellers</a>, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.<br />
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In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following <a href="#"pictures</a>.<br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(overalldescription)Team:Edinburgh/biology(overalldescription)2009-10-19T13:10:45Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Overall description</b></font><br />
<br />
<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<b> Personal note </b> <br /><br /><br />
Participating in the iGEM competition gives the opportunity to people to meet like-minded scientists around the world. The whole structure of the competition promotes the creation of a global scientific community, removing boundaries and making collaboration between different disciplines of science easier. Such a mentality is needed for tackling scientific issues of this era. Science has reached such a level where everyone has become <br />
so specialised, that sometimes the bigger picture is lost. Hopefully all of us can make a change for the future generations. Participating in iGEM inspired me in looking further into the beneficial prospects of synthetic biology and has motivated me to look further into this branch of science.<br />
<br /><br />
<font color="green" style="float:right"> Vasilis </font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/d/d8/Biology%28OverallDescription%29.jpg" style="margin-left:-5px;margin-top:60px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
Our project is concerned with the detection of both TNT and nitrites/ nitrates. We have designed two distinct pathways, each detecting the relevant substances. Via signal integration, both pathways interact to give a different visual signals depending on the presence or absence of our target chemicals. <br />
<br /><br /><br />
In this section, we present a simplified explanation of how our system operates from a practical point of view. The first aspect to point out is that we intend for the bacteria to be spread over affected areas with an aeroplane. The resulting colour pattern will be viewed at night. The visual outcome results are summarized in Table 1.<br />
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<br /><br /><br />
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<div id="left" style="width:50%"><br />
You will notice that this system has a limitation – When TNT is present in the absence of nitrite, there will be no visual outcome. This is because enhanced yellow fluorescent protein (EYFP) is produced in the presence of TNT and it is not excitable by visible light. However, in the presence of nitrite/ nitrates, luciferase will produce blue light that can excite EYFP. Adding a TNT degrading enzyme to our system solves this problem. The enzyme we have incorporated is PETN reductase, a nitroreductase enzyme from <i>Enterobacter cloacae</i> (Accession #: U68759, BBa_K216006). This enzyme will be produced in the presence of TNT and will degrade TNT to nitrites (French, 1998). Furthermore, TNT is degraded by naturally occurring soil flora and fauna (French, 1998). Hence, we are confident that TNT will always be present with nitrites in the soil.<br />
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<div id="right" style="width:49%"><br />
<div id="table" style="margin-left:15px;"<br />
<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th colspan="2"><center>Chemical</center></th><br />
<th rowspan="2"><center>Visual outcome</center></th><br />
</tr><br />
<tr><br />
<th><center>TNT</center></th><br />
<th><center>Nitrates/Nitrites</center></th><br />
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</tr><br />
<tr><br />
<td><center>Absent</center></td><br />
<td><center>Absent</center></td><br />
<td><center>No visual outcome</center></td><br />
</tr> <br />
<tr><br />
<td><b><center>Present</center></b></td><br />
<td><center>Absent</center></td><br />
<td><center>No visual outcome</center></td><br />
</tr> <br />
<tr><br />
<td><center>Absent</center></td><br />
<td><b><center>Present</center></b></td><br />
<td><font color="blue"><center>Blue light</center></font></td><br />
</tr> <br />
<tr><br />
<td><b><center>Present</center></b></td><br />
<td><b><center>Present</center></b></td><br />
<td><font color="#f7b60a"><center>Yellow light</center></font></td></tr> </table><br />
<i>Table 1. Visual outcome in different combinations of the detected chemicals</i><br />
</div><br />
</div><br />
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
<br />
<b>I am confused, what is the advantage of having two colours?</b> <br /><br /><br />
<br />
The colour pattern to be expected is quite simple- If you imagine TNT molecules diffusing away from their source of origin in all directions, you will realise that a pattern of concentric circles pinpointing the location of the landmine will be formed. As TNT is further away from the point of origin it becomes scarcer, and nitrites will predominate giving rise to a blue colour, whereas nearer the origin TNT will be in higher concentration giving rise to a yellow light. The predicted signal output in the presence of TNT is two concentric circles, blue on the outside, and yellow on the inside (Figure 1). If you would like more information on that, why don’t you check our <a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">modelling page</a>?<br />
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<br /><br /><br />
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<div id="left" style="width:50%"><br />
The two-colour system allows us to pinpoint the location of a landmine with a higher precision - imagine trying to pinpoint small areas of light in the night compared to looking for big areas of light with two different colours indicating the actual landmine position. More importantly, this two-colour system boasts a safety feature by creating a “buffer” zone in addition to indicating the exact position of a landmine. In this way, a person walking through a minefield in which the bacteria are spread will know when they are approaching a landmine long before reaching it. This prevents potential explosion of the mine.<br />
<br /><br /><br />
<b>Is it safe to spread synthetic bacteria on the soil?</b><br /><br /><br />
If you have concerns about biosafety or ethical issues, visit our <a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29">ethics</a> and <a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">biosafety</a> pages.<br />
</div><br />
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<div id="right" style="width:49%"><br />
<img src="https://static.igem.org/mediawiki/2009/4/4e/Visual-outcomes.jpg"><br />
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<center><i>Figure 1. Expected visual outcome of our system </i></center><br />
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<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(tntsensing)Team:Edinburgh/biology(tntsensing)2009-10-19T13:10:08Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
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</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:5px;"><b>Biology - TNT-sensing Pathway</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<b> Personal note </b> <br /><br /><br />
<br />
One of the reasons for studying biology for me has always been that scientific knowledge is power. The exhilarating and empowering IGEM experience has finally helped me understand that I really am one of the key holders to progress. <br /><br /> <br />
It has been said many times that science is not truly science unless it allows us to prove its concepts by creating things. Certainly for biology, the culmination and the peak of our knowledge will result in this ability, more precisely in the ability to apply engineering concepts to biological knowledge, to be able to demonstrate the facts through re-recreating them. I believe that to say that we are successfully doing this already would be an overstatement; nonetheless, I do believe that the steady path to our “masterhood” of biology has begun, and it is the taking part in this exciting, intriguing and perhaps mysterious new beginning that is the most rewarding part of being involved in IGEM. <br />
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<font color="green" style="float:right">Julia</font><br />
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The moment a landmine encounters soils, it leaks TNT (Jenkins et al., 2001). TNT will be detected by a computationally designed synthetic receptor, TNT.R1 (Looger et al., 2003). Since TNT.R1 is derived from ribose-binding protein and wild-type E. coli swims towards ribose, we hypothesize that E. coli cells expressing TNT.R1 will chemotaxi towards TNT. To verify this we modeled a chemotaxis experiment. To see the experimental results in detail please <a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">click here</a>.<br />
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Upon interaction with TNT, the receptor (BBa_K216002) will undergo a conformational change, allowing it to interact with Trg-EnvZ (a.k.a Trz) fusion protein (BBa_K216004). Previously, Trg-EnvZ was entered into the registry, unfortunately, the sequencing results were inconsistent. Furthermore, we failed to revive stabs received from the registry. As such, we contacted Dr Hazelbauer and colleges who kindly provided us with the plasmid carrying the fusion protein (Baumgartner et al., 1994).<br />
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Subsequently, Trg-EnvZ also undergoes conformational change and autophosphorylates. Eventually, it phosphorylates the secondary messenger ompR, and phosphorylated omp-R activates the ompC promoter (BBa_R0082).<br />
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Activation of PompC will result in upregulation of enhanced yellow fluorescent protein (eyfp) and onr. Onr encodes PETN reductase, a nitroreductase from Enterobactor cloacae (Accession #: U68759, BBa_K216006). PETN reductase will reduce TNT to nitrites (French et al., 1998) that would feed into the <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">nitrite/nitrate detection system.</a><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/79/TntSensingFigure1.jpg" height="370" width="500"></center><br /><br />
<i><b>Figure 1</b> TNT binds to TNT.R1 in the periplasm. The TNT-TNT.R1 complex induces a conformational change in the Trg-EnvZ (Trz) fusion protein. Trg-EnvZ autophosphorylates and subsequently phosphorylates ompR. Phosphorylated ompR activates transcription.</i><br />
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<img src="https://static.igem.org/mediawiki/2009/6/64/SignalIntegration.jpg" style="margin-left:-5px;margin-top:0px;"><br />
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You will also notice that EYFP is not excited by visible light, therefore no signal will be detected in the presence of TNT only. However, in the presence of nitrite, a blue-green light that will subsequently excite EYFP will be produced (Figure 2).<br />
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TNT.R1 and the Trg-EnvZ are preiplasmic and transmembrane proteins respectively. Therefore, high levels of expression can potentially disrupt the plasma membrane and result in cell death. Hence, we decided to express both proteins under the control of a weak consitutive promoter (BBa_J23105). This is the same promoter used to control expression of the genes coding for aldehyde-producing enzymes in the <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">nitrite/nitrate sensing system.</a><br />
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<img src="https://static.igem.org/mediawiki/2009/0/05/TNTSensingFigure2.jpg" height="280" width="480"><br />
<center><i><b>Figure 2</b> Signal integration.</i></center><br />
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The secondary messenger ompR and promoter PompC occur naturally in <i>E. coli</i>. Their function is to activate gene transcription in response to osmotic stress (Maeda et al., 1990). Additionally, it has been found that the PompC activity varies in response to procaine concentration. This finding led us to characterize PompC activity with varying procaine concentration. The characterization was done using Jason Kelly’s promoter characterization kit. <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Click here</a> for the results.<br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(nitritenitratesensing)Team:Edinburgh/biology(nitritenitratesensing)2009-10-19T13:04:19Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Nitrite/Nitrate-Sensing Pathway</b></font><br />
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<b> Personal note </b> <br /><br /><br />
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!<br />
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<font color="green" style="float:right">Evangeline</font><br />
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<img src="https://static.igem.org/mediawiki/2009/8/84/NitrateDetection.jpg" style="margin-left:-5px;margin-top:60px;"><br />
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TNT filled landmines often leak when it contact with the soil (Jenkins et al., 2001). The chemicals leaking from landmines include 1,3-DNB, 2,4-DNT, and 2,4,6-TNT (Jenkins et al., 2001). Subsequently, these chemicals are degraded to nitrites by soil bacteria (French et al., 1998). As such, we expect a radial diffusion gradients of nitrites around a focal point (the landmine). The size of this radius would depend on both the soil structure (e.g. water content, grain size etc) and the age of the landmine.<br />
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Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the <i>E. coli</i> yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (Bba_xxxx). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, <a href="https://2009.igem.org/Team:Edinburgh/biology(results)">click here</a>.<br />
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It is also noteworthy that the nitrite feeding into the nitrite-detecting pathway comes from two sources. First, nitrite that has been degraded by soil bacteria can enter the cell. Second, our E. coli features a nitroreductase (PETN reductase) that degrades TNT to nitrites within the cell (Figure 2).<br />
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In our system, PyeaR will control the expression of a fusion protein that combines <i>Photobacterium phosphoreum luciferase</i> (Accession #: AY341063, Bba_xxxx) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f7/PyeaRrepression.jpg" width="450"><br />
<center><i><b>Figure 1</b> a) PyeaR repression b) Repression relieved by nitrites. Transcription activated.</i></center><br />
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<img src="https://static.igem.org/mediawiki/2009/3/37/PETNReduces.jpg" width="550" height="350"><br />
<center><i><b>Figure 2</b> PETN reduces TNT to nitrite. Along with nitrite in the soil, it binds to the NsrR repressor to activate gene transcription. LuxAB.GFP is produced and blue-green light is emitted.</i></center><br />
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The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT detection</a>. <br />
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Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from <i>Xenorhabdus luminescens</i> (Bba_xxxx). The corresponding genes of <i>P. phosphoreum</i> were not used because they are expressed at very low quantities in <i>E. coli</i>. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in <i>E. coli</i> or condon usage bias (by <i>P. phosphoreum</i>). (Lee <i>et al.</i>, 2000 and Miyamoto et al., 1987).<br />
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After consulting our <a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">modellers</a>, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.<br />
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In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following <a href="#"pictures</a>.<br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(results)Team:Edinburgh/biology(results)2009-10-19T12:55:29Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Results</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal Note </b> <br /><br /><br />
</div><br />
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<img src="https://static.igem.org/mediawiki/2009/7/71/OmpcCharachterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/1/13/Ompc-promoter.jpg"> </center><br />
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We used Jason Kelly's promoter characterization kit to characterize the <i>ompC</i> promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.<br />
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<img src="https://static.igem.org/mediawiki/2009/b/bb/YeaRPRomoterCharacterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<b> Miller Test </b> <br /><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/e/ef/Millertest.jpg"> </center> <br /><br /><br />
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The classical Miller's Assay was used to characterize the <i>yeaR</i> promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.<br />
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<br /><br /><br />
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<b> Jason Kelly Assay </b> <br /><br />
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<center> <img src="https://static.igem.org/mediawiki/2009/d/d8/Jasonkelly.jpg"> </center> <br /><br /><br />
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From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.<br />
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</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/8/8d/EdinburghFurtherWork.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed! <br />
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<br /><br /><br />
<br />
<b>Testing TNT receptors</b><br /><br /><br />
<br />
First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.<br />
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<br /><br /><br />
<br />
<b>Environmental aspect</b> <br /><br /><br />
<br />
For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.<br />
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<b>Field test</b> <br /><br /><br />
<br />
Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient. <br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(results)Team:Edinburgh/biology(results)2009-10-19T12:50:43Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Results</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
<br />
<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal Note </b> <br /><br /><br />
</div><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/7/71/OmpcCharachterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<br /><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2009/1/13/Ompc-promoter.jpg"> </center><br />
<br />
<br /><br /><br />
<br />
We used Jason Kelly's promoter characterization kit to characterize the <i>ompC</i> promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.<br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/b/bb/YeaRPRomoterCharacterisation.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
<br /><br />
<b> Miller Test </b> <br /><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2009/e/ef/Millertest.jpg"> </center> <br /><br /><br />
<br />
The first test done on the YeaR promoter was the famous miller test. As you can see this test shows that pYeaR is more sensitive to Nitrates than to Nitrites, and the sensitivity increases with increased concentration. In order to get a more detailed representation to the response to nitrates, the main activator of this promoter, the Jason Kelly assay was performed.<br />
<br />
<br /><br /><br />
<br />
<b> Jason Kelly Assay </b> <br /><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2009/d/d8/Jasonkelly.jpg"> </center> <br /><br /><br />
<br />
From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.<br />
<br />
</div><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/8/8d/EdinburghFurtherWork.jpg" style="margin-left:-5px;margin-top:20px;"><br />
<br />
<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
<br />
After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed! <br />
<br />
<br /><br /><br />
<br />
<b>Testing TNT receptors</b><br /><br /><br />
<br />
First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.<br />
<br />
<br /><br /><br />
<br />
<b>Environmental aspect</b> <br /><br /><br />
<br />
For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.<br />
<br />
<br /><br /><br />
<br />
<b>Field test</b> <br /><br /><br />
<br />
Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient. <br />
<br />
</div><br />
</div><br />
<div id=bottombox><br />
<div id=bottomboxtext align=center><br />
<font size=2>Edinburgh University iGEM Team 2009</font> <br />
</div><br />
</div><br />
<br />
</div> <br />
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</div><br />
</body><br />
</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T12:44:46Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
<br />
<div id=Edinburghcontent><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
<br />
<br /><br /><br />
<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
</div><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (BBa_K216002 & BBa_K216003)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> (BBa_K216004)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a> (BBa_R0082)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm4.static.flickr.com/3504/4025313507_0097490319.jpg"></td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T12:43:40Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (BBa_K216002 & BBa_K216003)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> (BBa_K216004)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a> (BBa_R0082)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
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Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
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</td><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
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</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td><img src="http://farm3.static.flickr.com/2790/4026052632_7e7dd79dbe.jpg"></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
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</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
</div><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/team(teammembers)Team:Edinburgh/team(teammembers)2009-10-19T11:29:03Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Team Members</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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Here we are! Edinburgh University 2009 iGEM Team :P This our our video team introduction. Every single person who had impact on our team project has his own small video snippet. Check this out!<br /><br />
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<img src="https://static.igem.org/mediawiki/2009/f/fa/TeamBiologists.jpg" style="margin-left:-5px;margin-top:10px;"><br />
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<img src="https://static.igem.org/mediawiki/2009/4/42/Vas.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Vasilis Andreou <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hey guys my name is Vasilis Andreou. Something you don't know about me is that my house in Nicosia, Cyprus is about 2 &nbsp;&nbsp;&nbsp;minutes from the borders dividing the town, and in the opposite direction is about 2 minutes from the country's prison. See you &nbsp;&nbsp;&nbsp;in MIT!!<br /><br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>V.Andreou<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Evangeline JiaLing Oh <br /><br /><br />
&nbsp;&nbsp;&nbsp;I was born and bred in Singapore. Apart from ice-cream, I love gene regulatory networks. Sometime in the near future, I hope &nbsp;&nbsp;&nbsp;to be BioBricking in eukaryotic cells. Have fun and break a leg in MIT.<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>E.J.Oh-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/b/b8/EdinburghDasha%28edited%29.jpg" width="150" height="150" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Dasha <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hi I am Dasha and I love biology. iGEM has been the most exhilarating and challenging<br />
project I have ever done, and it made me &nbsp;&nbsp;&nbsp;realize how many possibilities are presented to<br />
me by what I study! <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>D.Ausiannikava<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c5/Juliaedinburgh.jpg" width="150" height="280" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Julia <br /><br /><br />
&nbsp;&nbsp;&nbsp;My name is Julia and I was born in Kiev, Ukraine. I moved to Belgium at a young age and since then I have had the pleasure of &nbsp;&nbsp;&nbsp;being a citizen of the world, having lived in Ireland, UK and the Middle East—and the list keeps growing! I have degrees in &nbsp;&nbsp;&nbsp;Biotechnology and Drug Discovery, and particularly love everything gene-related. <br /><br /><br />
&nbsp;&nbsp;&nbsp;To me, IGEM conveys an emotion. A mixture of excitement, uncertainty and, most intriguingly, empowerment. If you, as a &nbsp;&nbsp;&nbsp;biologist, ever doubt the practical potential of your knowledge, I challenge you to take part in this competition—you will &nbsp;&nbsp;&nbsp;instantly understand what kind of powerful sci-fi stuff lurks within your brain! <br />
<br /><br />
<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>J.Skripka<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/1/1b/TeamModellers.jpg" style="margin-left:-5px;margin-top:10px;"><br />
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<img src="https://static.igem.org/mediawiki/2009/d/db/WinyEdinburgh.jpg" width="150" height="281" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Winston Ho <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello there! Names Winston, born in the 'deen, which is up in the North-East of Scotland. I'm studying chemical engineering, &nbsp;&nbsp;&nbsp;having just started my 4th year at Edinburgh. Can't wait to join the oil industry ;) I also have a soft spot for biology =) CU at MIT!<br />
<br /><br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b> W.Ho-3<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f5/Rachel.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rachel "Lady GaGa" Morrison <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello, my name is Rachel though over the course of the summer my lovely team mates have called me a variety of things &nbsp;&nbsp;&nbsp;including Lady Gaga. I am currently in my final year of a master’s of Chemical Engineering though I have spent most of the &nbsp;&nbsp;&nbsp;summer learning how to model gene expression networks, program in JAVA and MATLAB and have also picked up some &nbsp;&nbsp;&nbsp;biology along the way! <br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>R.A.Morrison-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<b> &nbsp;&nbsp; Name: </b> JD <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email:</b> jan.domozilov<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rohin <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<b> &nbsp;&nbsp; Name: </b> Sam Kammerling<br />
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&nbsp;&nbsp;&nbsp;I'm Sam, and I have been singing theme tunes for scientists for as long as I can <br />
remember. Growing up in the English &nbsp;&nbsp;&nbsp;countryside it was every child's dream to have your <br />
voice heard on the international scientific stage, and now this dream has &nbsp;&nbsp;&nbsp;come true <br />
thanks to the Edinburgh iGem team. Singing about bacteria is my passion, and I hope I <br />
inspire others to be as &nbsp;&nbsp;&nbsp;passionate about bacteria as me. Big love peeps x<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c3/KacperEdinburgh.jpg" width="150" height="227" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Kacper Rogala <br /><br /><br />
&nbsp;&nbsp;&nbsp;I study Biochemistry, love film and good food! I was engaged to film, act in and produce the team's awesome promotional &nbsp;&nbsp;&nbsp;video. This picture of me was taken during the "explosion scene". I am also a hairy chewbacca.<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br />
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We are very international team, so we thought it might be interesting for you to know bit more about places we all came from. Our team consists of 8 undergraduates from 7 different countries. <br /><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/team(teammembers)Team:Edinburgh/team(teammembers)2009-10-19T11:27:54Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29"> Team Members </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29"> Advisors </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29"> Supervisors </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28edinburghuniversity%29"> Edinburgh University </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28our%20message%29"> Our message </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29"> Gallery </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29"> Contacts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29"> Acknowledgements </a> </div><br />
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<ul id="nav" class="dropdown dropdown-horizontal"><br />
<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Team - Team Members</b></font><br />
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Here we are! Edinburgh University 2009 iGEM Team :P This our our video team introduction. Every single person who had impact on our team project has his own small video snippet. Check this out!<br /><br />
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<b> &nbsp;&nbsp; Name: </b> Vasilis Andreou <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hey guys my name is Vasilis Andreou. Something you don't know about me is that my house in Nicosia, Cyprus is about 2 &nbsp;&nbsp;&nbsp;minutes from the borders dividing the town, and in the opposite direction is about 2 minutes from the country's prison. See you &nbsp;&nbsp;&nbsp;in MIT!!<br /><br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>V.Andreou<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br /><br /><br /><br />
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<b> &nbsp;&nbsp; Name: </b> Evangeline JiaLing Oh <br /><br /><br />
&nbsp;&nbsp;&nbsp;I was born and bred in Singapore. Apart from ice-cream, I love gene regulatory networks. Sometime in the near future, I hope &nbsp;&nbsp;&nbsp;to be BioBricking in eukaryotic cells. Have fun and break a leg in MIT.<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>E.J.Oh-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<b> &nbsp;&nbsp; Name: </b> Dasha <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hi I am Dasha and I love biology. iGEM has been the most exhilarating and challenging<br />
project I have ever done, and it made me &nbsp;&nbsp;&nbsp;realize how many possibilities are presented to<br />
me by what I study! <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b>D.Ausiannikava<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/c/c5/Juliaedinburgh.jpg" width="150" height="280" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Julia <br /><br /><br />
&nbsp;&nbsp;&nbsp;My name is Julia and I was born in Kiev, Ukraine. I moved to Belgium at a young age and since then I have had the pleasure of &nbsp;&nbsp;&nbsp;being a citizen of the world, having lived in Ireland, UK and the Middle East—and the list keeps growing! I have degrees in &nbsp;&nbsp;&nbsp;Biotechnology and Drug Discovery, and particularly love everything gene-related. <br /><br /><br />
&nbsp;&nbsp;&nbsp;To me, IGEM conveys an emotion. A mixture of excitement, uncertainty and, most intriguingly, empowerment. If you, as a &nbsp;&nbsp;&nbsp;biologist, ever doubt the practical potential of your knowledge, I challenge you to take part in this competition—you will &nbsp;&nbsp;&nbsp;instantly understand what kind of powerful sci-fi stuff lurks within your brain! <br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br />
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<b> &nbsp;&nbsp; Name: </b> Winston Ho <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello there! Names Winston, born in the 'deen, which is up in the North-East of Scotland. I'm studying chemical engineering, &nbsp;&nbsp;&nbsp;having just started my 4th year at Edinburgh. Can't wait to join the oil industry ;) I also have a soft spot for biology =) CU at MIT!<br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b> W.Ho-3<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f5/Rachel.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rachel "Lady GaGa" Morrison <br /><br /><br />
&nbsp;&nbsp;&nbsp;Hello, my name is Rachel though over the course of the summer my lovely team mates have called me a variety of things &nbsp;&nbsp;&nbsp;including Lady Gaga. I am currently in my final year of a master’s of Chemical Engineering though I have spent most of the &nbsp;&nbsp;&nbsp;summer learning how to model gene expression networks, program in JAVA and MATLAB and have also picked up some &nbsp;&nbsp;&nbsp;biology along the way! <br />
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<b> &nbsp;&nbsp;&nbsp;Contact email: </b>R.A.Morrison-2<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">sms.ed.ac.uk<br />
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<img src="https://static.igem.org/mediawiki/2009/4/42/Vas.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> JD <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email:</b> jan.domozilov<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/88/At_sign.svg/145px-At_sign.svg.png" height="20" width="20">gmail.com<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/TeamEvan.jpg" width="150" height="230" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Rohin <br /><br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8b/TeamGueststars.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<img src="https://static.igem.org/mediawiki/2009/c/cf/Sam_Kammerling.jpg" width="150" height="200" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Sam Kammerling<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;I'm Sam, and I have been singing theme tunes for scientists for as long as I can <br />
remember. Growing up in the English &nbsp;&nbsp;&nbsp;countryside it was every child's dream to have your <br />
voice heard on the international scientific stage, and now this dream has &nbsp;&nbsp;&nbsp;come true <br />
thanks to the Edinburgh iGem team. Singing about bacteria is my passion, and I hope I <br />
inspire others to be as &nbsp;&nbsp;&nbsp;passionate about bacteria as me. Big love peeps x<br />
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<br /><br />
<br /><br />
<br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/c/c3/KacperEdinburgh.jpg" width="150" height="227" align="left"><br />
<b> &nbsp;&nbsp; Name: </b> Kacper Rogala <br /><br /><br />
&nbsp;&nbsp;&nbsp;I study Biochemistry, love film and good food! I was engaged to film, act in and produce the team's awesome promotional &nbsp;&nbsp;&nbsp;video. This picture of me was taken during the "explosion scene". I am also a hairy chewbacca.<br />
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<br /><br />
<b> &nbsp;&nbsp;&nbsp;Contact email: </b><br />
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We are very international team, so we thought it might be interesting for you to know bit more about places we all came from. Our team consists of 8 undergraduates from 7 different countries. <br /><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T11:13:20Z<p>Evangelineoh: </p>
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<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29"> Overall Description & Design </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29"> TNT-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29"> Nitrite/Nitrate-Sensing Pathway</a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29"> Biobrick Parts </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29"> Results </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29"> Problem Solving and Tips </a> </div> <br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29"> Materials and Methods </a> </div><br />
<div id=menuitem > <a href="https://2009.igem.org/Team:Edinburgh/biology%28references%29"> References </a> </div><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29" class="dir">Biology</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28overalldescription%29">Overall Description and Design</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">TNT-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28nitritenitratesensing%29">Nitrite/Nitrate-Sensing Pathway</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">Biobrick Parts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28solvedproblems%29">Problem Solving and Tips</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/biology%28materialsandmethods%29">Materials and Methods</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29" class="dir">Modelling</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28overalldescription%29">Overall Description</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
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<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
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<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_K216009) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (BBa_K216002 & BBa_K216003)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> (BBa_K216004)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a> (BBa_R0082)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
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<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(BBa_K216009)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
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</html></div>Evangelineohhttp://2009.igem.org/Team:Edinburgh/biology(biobricks)Team:Edinburgh/biology(biobricks)2009-10-19T11:12:38Z<p>Evangelineoh: </p>
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<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28generegulatorynetwork%29">Gene Regulatory Network</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28reallifemodelling%29">Real Life Modelling</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/reallifeapplication%28scaleup%29">Scale Up</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28results%29">Results</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/modelling%28references%29">References</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28introduction%29" class="dir">Underlying Philosophy</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28publicperception%29">Public Perception</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28legislationissues%29">Legislation issues</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28biosafety%29">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/ethics%28religiousperception%29">Religious Perception</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29" class="dir">Informatics</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28introduction%29">Introduction</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conceptsandtechnology%29">Concepts and Technologies</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28globetutorial%29">Globe Tutorial</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28igemwikhacks%29">iGEM WIKI Hacks</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/newinformatics%28conclusions%29">Conclusions</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/Notebook">Notebook</a></li><br />
<br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teamintroduction%29" class="dir">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28teammembers%29">Team Members</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28advisors%29">Advisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28supervisors%29">Supervisors</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28gallery%29">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28contacts%29">Contacts</a></li><br />
<li><a href="https://2009.igem.org/Team:Edinburgh/team%28acknowledgements%29">Acknowledgements</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<font color="#323131" style="font-size:14px;float:left;margin-left:20px;margin-top:20px;"><b>Biology - Biobrick Parts</b></font><br />
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<div id=Edinburghcontent><br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/EdinburghPagemarker.jpg" style="margin-left:766px;"><br />
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<div id="abstarct" style="background-color:#e4f5ca;border:1px solid #595151;width:755px;height:180px;margin-top:10px;margin-left:20px;font-size:11px;text-align:justify;padding-left:5px;padding-right:5px;padding-top:5px;padding-bottom:5px;"><br />
<b> Personal note </b> <br /><br /><br />
We worked really hard over this summer, but don't think it was all work and no play. One<br />
of the highlights was making our promotional video clip. If you haven't already seen it<br />
check it out and it will hopefully make your day! For other activities check our Gallery!<br />
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<font color="green" style="float:right">Vasilis</font><br />
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</div><br />
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<img src="https://static.igem.org/mediawiki/2009/5/5f/BiobricksInfo.jpg" style="margin-left:-5px;margin-top:20px;"><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<div id="left" style="width:38%;padding-bottom:45px;height:320px;"><br />
<b>Quick links:</b> <br /><br /><br />
<a href="#tnt.r1">tnt.R1</a> (BBa_K216002)<br /><br />
<a href="#tnt.r3">tnt.R3</a> (BBa_K216003)<br /><br />
<a href="#fusion">trz fusion protein</a> (BBa_K216004) <br /><br />
<a href="#ompc">ompC Promoter</a> (BBa_R0082) <br /><br />
<a href="#enhanced">Enhanced Yellow Fluorescent Protein</a> (BBa_E0430)<br /><br />
<a href="#onr">onr</a> (BBa_K216006) <br /><br />
<a href="#year">yeaR promoter</a> (BBa_K216005) <br /><br />
<a href="#luxab">luxAB-gfp fusion protein </a>(BBa_xxxx) <br /><br />
<a href="#lump">lump</a> (BBa_K216007) <br /><br />
<a href="#luxcde">luxCDE from <i>X. luminescence</i></a> (BBa_xxxx) <br /><br />
<a href="#nsrr">nsrR from Nitrosomonas Europaea</a> (BBa_xxxx) <br /><br />
<a href="#constitutive">Constitutive promoter </a> (BBa_J23105) <br /><br />
<a href="#pompc">PompC-eyfp-onr</a> (BBa_K216012) <br /><br />
<a href="#pompcgfp">PompC-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pompceyfp">PompC-eyfp</a> (BBa_K216011) <br /><br />
<a href="#pompclacz">PompC-lacZ’</a> (BBa_K216010) <br /><br />
<a href="#pyeargfp">PyeaR-GFP</a> (BBa_xxxx) <br /><br />
<a href="#pyearlacz">PyeaR-lacZ’</a> (BBa_xxxx) <br /><br />
<a href="#pconstitutive">Pconstitutive- tnt.r1-trz</a> (BBa_xxxx) <br /><br />
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<div id="right" style="width:61%;padding-bottom:75px;height:220px;"><br />
<b>Key</b> <br /><br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg" height="50" width="50"> - new BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg" height="50" width="50"> - existing BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg" height="50" width="50"> - hypothethical BioBrick<br />
&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png" height="50" width="50"> - composite BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg" height="50" width="50"> - fixed BioBrick<br /><br />
<img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg" height="50" width="50"> - characterized BioBrick<br /><br />
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</div><br />
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<div id="text" style="margin-left:20px;margin-top:10px;padding-bottom:15px;"><br />
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<table cellspacing="0" cellpadding="0"><br />
<tr><br />
<th><center>Biobrick Name</center></th><br />
<th><center>Key</center></th><br />
<th><center>Description</center></th><br />
</tr><br />
<tr><br />
<td><center><b><a name="tnt">tnt.R1 & tnt.R3</a> (BBa_K216002 & BBa_K216003)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>TNT.R1 and TNT.R3 are computationally derived ligand receptors specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that each receptor recognizes TNT instead of the wild-type ligand.<br />
<br /><br /><br />
<font style="font-size:10px">Looger, L. L., Dwyer, M. A., Smith J. J., and Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185-190 (2003).</font></td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="fusion">trz fusion protein</a> (BBa_K216004)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/e/e3/FixedBioBrick.jpg"></center></td><br />
<td>This hybrid protein contains the periplasmic and transmembrane domains of the chemoreceptor Trg and the cytoplasmic domain of the osmosensor EnvZ. Upon interaction with the TNT-TNT.R1/R3 complex, Trg-EnvZ undergoes a conformational change and autophosphorylates. Subsequently, it phosphorylates the second messenger ompR.<br />
<br /><br /><br />
This part was previously submitted into the Registry (BBa_J58104). However, the sequence information is inconsistent. We requested stabs from the Registry, but failed to obtain the correct construct. Hence, we contacted the following authors and they kindly sent us plasmids carrying the construct.<br />
<br /><br /><br />
<font style="font-size:10px">Baumgartner, J. W., Kim, C., Brisette, R. E., Inoue, M., Park, C., and Hazelbauer, G. L. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognises sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of Bacteriology, 1157-1163 (1994).</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="ompc">ompC promoter</a> (BBa_R0082)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>The <i>E. coli</i> EnvZ-OmpR two-component system is a well-characterized signaling pathway. EnvZ, a membrane protein, regulates the levels of phosphorylated OmpR (OmpR-P), which in turn regulates gene transcription. The best-studied genes regulated by this system are <i>ompF</i> and <i>ompC</i>. There are several OmpR binding sites at the <i>ompF</i> and <i>ompC</i> promoters.<br />
<br /><br /><br />
In our project, a fusion protein (Trg-EnvZ, J58104) carrying the EnvZ histidine kinase activity will phosphorylate ompR in response to TNT binding to a receptor. ompR-P will bind to the upstream binding sequences, thereby controlling genes of interest. The genes that we have chosen to put under the control of the ompR-controlled promoter are those coding for enhanced yellow fluorescent protein and PETN reductase <i>(onr)</i>.<br />
<br /><br /><br />
It is believed that the native EnvZ-OmpR senses changes in osmolarity. High osmolarity activates EnvZ, thereby generating more ompR-P that binds to the upstream operator sites of ompC. However, in 2006, Batchelor and Goulian compared the effects of osmolarity and procaine and concluded that procaine activates EnvZ-OmpR signaling whereas osmolarity only has a weak effect on the system.<br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Batchelor, E., and Goulian, M. Imaging OmpR localization in <i>Escherichia coli</i>. Molecular Microbiology 59(6),1767-78 (2006).<br />
<br /><br /><br />
Maeda, S., and Mizuno T. Evidence for multiple Omp-R binding sites in the upstream activation sequence of the ompC promoter in <i>Escherichia coli</i>: a single OmpR-binding site is capable of activating the promoter. J. Bacteriol. 172 (1), 501-503 (1990).<br />
</font><br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="enhanced">enhanced yellow fluorescent protein</a> (BBa_E0430)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>In our system, EYFP is expressed in the presence of TNT and under the control of the ompC promoter (BBa_R0082). The protein is excited by light emitted by the LuxAB.GFP fusion protein that is produced in the presence of nitrites.<br />
</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="onr">onr</a> (BBa_K216006)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td><i>onr</i> codes for PETN (pentaerythritol tetranitrate) reductase that degrades components of landmines.<br />
PETN reductase was cloned from <i>Enterobacter</i> cloacae PB2, a strain isolated from explosive-contaminated soil (Binks, 1996). PETN reductase is a small monomeric flavoprotein that reduces a wide variety of electron-deficient multi-nitro compounds including pentaerythritol tetranitrate (PETN), nitroglycerine (glycerol trinitrate, GTN), ethylene glycol dinitrate (EGDN), and trinitrotoluene (TNT). In the case of nitrate esters, the products are nitrite and alcohol; in the case of nitroaromatics such as TNT, the initial product is a hydrode adduct (hydride-Meisenheimer complex), which is further reduced to the dihydride adduct. This then degrades in an unknown way with liberation of nitrite and non-aromatic products. <br />
<br /><br /><br />
<font style="font-size:10px"><br />
Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219 (1996).</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="year">yeaR promoter</a> (BBa_K216005)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"><br /><img src="https://static.igem.org/mediawiki/2009/b/be/CharacterizedBio.jpg"></center></td><br />
<td>Microarray studies have identified the yeaR-yoaG operon among genes whose transcription is induced in response to nitrate, nitrite or nitric oxide. <br />
<br /><br /><br />
Nitrate and nitrite regulate gene expression in anaerobic conditions via the two-component systems NarX-NarL and NarQ-NarP. Typically, Nar-activated genes depend on the oxygen-responsive Fnr transcription activator. However, the transcription from the year-yoaG operon is independent of Fnr activation. This makes the upstream regulatory sequence a good candidate for use as a nitrite-sensor in aerobic conditions. Nitrite is a product of natural TNT degradation. Elevated levels of environmental nitrite will indicate the presence of TNT.<br />
<br /><br /><br />
The response to nitrate and nitrite is regulated through the Nar regulatory system while response to nitric oxide acts through the NsrR repressor. The binding sites for phospho-NarL and –NarP activators and that for the NsrR repressor overlap in a region 62 nt upstream of the transcription initiation site. <br />
<br /><br /><br />
<a href="#">Click here</a> for the characterization results for this promoter.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Lin, H. Y., Bledsoe, P.J., and Stewart V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in <i>Escherichia coli</i> K-12. J Bacteriol. 189(21),7539-48 (2007).<br />
</font><br />
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</td><br />
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</tr><br />
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<tr><br />
<td><center><b><a name="luxab">luxAB-gfp fusion protein </a> (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td><i>Photobacterium phosphoreum</i> is a luminescent marine bacterium. It has the ability to produce light via the action of a luciferase enzyme. This enzyme is a heterodimer constituting two homologues, LuxA and LuxB. The reaction catalyzed by luciferase is shown below:<br />
<br /><br /><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Luciferase<br /><br />
RCHO + FMNH2+ O2 ------------------> RCOOH + FMN + H2O + light<br />
<br /><br /><br />
where R is the alkyl residue, FMNH2 is reduced flavin mononucleotide, and FMN is the flavin mononucleotide. <br />
<br /><br /><br />
The aldehyde substrate for the reaction can be produced by enzymes encoded by luxCDE (see below).<br />
<br /><br /><br />
The reason why Photobacterium phosphoreum luciferase was chosen over other luciferases is because the light it emits is relatively more intense <a href="#">(see picture)</a>. The wavelength of light emitted is λ max 478 nm. <br />
<br /><br /><br />
We have decided to make a LuxAB.GFP fusion protein as we hypothesize that this will increase the intensity of light. This hypothesis is founded upon an observation made by Miyawaki. Miyawaki created a fusion protein comprising of coelenterazine luciferase and yellow fluorescent protein. This resulted in a 7-fold increase in luminescence of the construct. We adopted GFP in our system as the activation spectrum closely matches the emission spectrum of <i>Photobacterium phosphoreum</i> luciferase.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Mancini, J. A., Boylan, M., Soly, R. R., Graham, A. F., and Meighen, E. A. Cloning and Expression of the <i>Photobacterium phosphoreum</i> Luminescence System Demonstrates a Unique lux Gene Organization. The Journal of Biological Chemistry 263 (28), 14308-14314 (1988).<br />
<br /><br /><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 84, 8912-8916 (1987). <br />
<br /><br /><br />
Miyawaki, A., Bringing bioluminescence into the picture. Nature Methods 4, 616 - 617 (2007).<br />
</font><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="lump">lump </a>(BBa_K216007)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>LumP encodes for lumazine protein. Lumazine interacts with the <i>Photobacterium phosphoreum</i> luciferase and shift the emission peak from 478 nm to 495 nm. This is crucial for our project because the blue light emitted by luciferase is required to activate the Aequorea victoria GFP.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Daubner, S. C., Astorga, A. M., Leisman, G. B. & Baldwin, T. O. Yellow light emission of vibrio fischeri strain y-1: purification and characterization of the energy-accepting yellow fluorescent protein. <i>Proceedings of the National Academy of Sciences of the United States of America</i> 84, 8912-8916 (1987).<br />
<br /><br /><br />
O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. C. Borrowed proteins in bacterial bioluminescence. <i>Proc Natl Acad Sci U S A</i>. 88(4), 1100–1104 (1991).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="luxcde">luxCDE</a> from X. luminescence (Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/NewBioBrick.jpg"></center></td><br />
<td>The <i>P. phosphoreum</i> genes coding for enzymes that produce aldehydes required for the luciferase reaction are expressed at very low levels in E. coli. As such, we adopted the aldehyde producing system from X. luminescens.<br />
<br /><br /><br />
The reasons for this low expression are not clear, but it could be due to the presence of repressors in E. coli or the usage of codons (by P. phosphoreum) where corresponding t-RNas are of very low abundance in E. coli. As both luciferases are able to use decanal (C9H19CHO) as a substrate, we anticipate that luciferase from P. phosphoreum will be able to use the aldehyde produced by X. luminescens’ s enzymes.<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Colepicolo, P., Cho, K. W., Poinar, G. O., and Hastings, J.W. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl Environ Microbiol. 55(10), 2601–2606 (1989).<br />
<br /><br /><br />
Lee, C. Y., and Meighrn, E. A. Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from <i>Photobacterium phosphoreum</i>. The Journal of Microbiology. 38(2), 80-87 (2000).<br />
<br /><br /><br />
Miyamoto, C., Byers, D., Graham, A. F., and Meighen E. A. Expression of bioluminescence by <i>Escherichia coli</i> Containing Recombinant Vibrio harveyi DNA. Journal of Bacteriology. 169(1), 247-253 (1987).<br />
<br />
</font><br />
</td><br />
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</tr><br />
<br />
<tr><br />
<td><center><b><a name="nsrr">nsrR from <i>Nitrosomonas Europaea</i></a>(BBa_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>NsrR was first described as a nitrite-responsive regulator of the nirK gene encoding nitrite reductase in <i>Nitrosomonas europaea</i> (Beaumont et al., 2004). NsrR sensitivity to nitrite was increased under acid conditions, so there is the possibility that NsrR is inactivated by the NO formed enzymatically as a by-product of nitrate and nitrite reduction or by disproportionation (Spiro, 2007), but no experimental evidence supporting the latter observation has been received up to now.<br />
<br />br /><br />
NsrR orthologues belong to the wider Rrf2 family of transcriptional repressors. The best characterized member of the family is the E. coli IscR protein, which contains a <a href="#">2Fe-2S</a> cluster. IscR has only three cysteine residues, which presumably provide three of the ligands to the <a href="#">Fe-S</a> luster. These cysteines are conserved in NsrR (with some variation in spacing) so it has been suggested that NsrR contains an <a href="#">Fe-S</a> cluster (Spiro, 2007).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
<br /><br /><br />
Spiro, S. Regulators of bacterial responses to nitric oxide. FEMS Microbiology Reviews 31, 193-211, (2007).<br />
</font><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="nirk">nirK promoter </a>(BBa_xxxx) </b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/f/fb/HypothethicalBio.jpg"></center></td><br />
<td>The repressor described above (NsrR) binds to this promoter in the absence of nitrites thus preventing transcription of the luciferase gene downstream. In the presence of nitrites the repressor releases from DNA and allows transcription of the luciferase gene. Thus in nitrite-rich medium the cells will be able to make blue light. <br />
<br /><br /><br />
This promoter is in the intergeneric region between the gene encoding the repressor NsrR (<b>yhdE</b>, NE0928 in the genome sequence, which is divergently transcribed), and the gene <b>'pan'</b> (NE0927) in the genome sequence (Beaumont et al., 2004).<br />
<br /><br /><br />
<font style="font-size:10px"><br />
Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M., Westerhoff, H. V., and Spanning, R. J. M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology 54 (1), 148-158, 2004.<br />
</font><br />
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</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="constitutive">Constitutive promoter</a>(BBa_J23105)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/5/52/ExisitingBio.jpg"></center></td><br />
<td>This will control the expression of tnt.r1, trz and luxCDE genes. As TNT.R1 is a periplasmic protein and Trz is a transmembrane protein, elevated amounts of these protein can potentially disrupt the cell membrane. Hence, we chose a weak constitutive promoter.<br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompc">PompC-eyfp-onr</a>(BBa_K216012)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Final construct in system.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompcgfp">PompC-GFP </a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240). Used for Jason Kelly’s promoter characterization kit.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompceyfp">PompC-eyfp</a>(BBa_K216011)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pompclacz">PompC-lacZ’</a>(BBa_K216010)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyeargfp">PyeaR-GFP</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PompC (Bba_R0082) and GFP (Bba_E0240)</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pyearlacz">PyeaR-lacZ’</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><center><b><a name="pconstitutive">Pconstitutive- tnt.r1-trz</a>(Bba_xxxx)</b></center></td><br />
<td><center><img src="https://static.igem.org/mediawiki/2009/c/c1/CompositeBioBrick.png"></center></td><br />
<td>Construct carrying PyeaR (BBa_K216005) and lacZ’ (Bba_J33202). Used for Miller’s assay.</td><br />
<br />
</tr><br />
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</table><br />
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<font size=2>Edinburgh University iGEM Team 2009</font> <br />
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