Team:Exeter/Parts

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<div id="toctitle"><h2>Contents</h2></div>
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<li class="toclevel-1"><a href="#1"><span class="tocnumber">1.</span> <span class="toctext">Our Parts</span></a></li>
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<li class="toclevel-1"><a href="#2"><span class="tocnumber">2.</span> <span class="toctext">Basic Parts</span></a></li>
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<li class="toclevel-1"><a href="#3"><span class="tocnumber">3.</span> <span class="toctext">Composite Parts</span></a></li>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
 
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<li class="toclevel-1"><a href="#TheNemRrecognitionpromoter(BBa_K1398008)"><span class="tocnumber">1</span> <span class="toctext">The NemR recognition promoter (BBa_K1398008)</span></a>
 
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<li class="toclevel-1"><a href="#TheNemRpromoterconstruct(BBa_K1398007)"><span class="tocnumber">1.1</span> <span class="toctext">The NemR promoter construct (BBa_K1398007)</span></a></li>
 
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<li class="toclevel-1"><a href="#References"><span class="tocnumber">2</span> <span class="toctext">References:</span></a></li>
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<h2> <span class="mw-headline" id="TheNemRrecognitionpromoter(BBa_K1398008)">The NemR recognition promoter (<a href="http://parts.igem.org/Part:BBa_K1398008">BBa_K1398008</a>)</span></h2>
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<p> One of the problems faced by the 2009 Edinburgh iGEM team was the issue of how TNT was to be detected in order to generate a gene expression response.</p>
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<a href="https://2009.igem.org/Team:Edinburgh/biology%28results%29">https://2009.igem.org/Team:Edinburgh/biology%28results%29</a>
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<a href="https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29">https://2009.igem.org/Team:Edinburgh/biology%28biobricks%29</a>
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<p>While their design and parts demonstrated a staggering amount of work and understanding, we felt that their proposed TNT-sensing construct was too complex and could be simplified. As a system of parts we felt that it relied too heavily on several freshly designed and uncharacterised parts working together in unison to be easily engineered for success. </p>
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<img src="https://static.igem.org/mediawiki/2014/8/83/Exeter_Edinburgh_TNT.jpg" style="margin-right: 200px; margin-left: 200px;" alt="NemA construct">
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<p align="center"><i><strong>Figure 7: </strong>TNT sensing pathway developed by the Edinburgh 2009 iGEM team.
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<a href="https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29">https://2009.igem.org/Team:Edinburgh/biology%28tntsensing%29</a>
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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></p>
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<p>&nbsp;</p>
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<p>As a result, we decided to see if we could discover and develop a simpler system that would be easier to engineer and conceptually extrapolate to other chemicals. After the discovery that the NemA enzyme could already be expressed at a very low level in <i>E.coli</i>, it was hypothesised that ''E.coli'' should have a gene regulatory molecule which controlled the nemA operon enabling its expression only in environments where TNT was present. In <i>E. coli</i>, nemA is located downstream of a gene encoding a transcription factor known as NemR, previously entitled YdhM29.  Disruption to the gene encoding NemR results in a decrease in nemA expression, both in the basal and induced states, which indicated that the nemA gene and NemR form a single, connected operon. Umezawa et al. (2008) proposed that the function of NemR is as a redox-operated transcriptional repressor of the nemA gene.
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NemR is proposed to remain bound to the operon and, when it binds TNT in the environment, undergo a conformational change to expose the promoter. The region of the promoter sequence to which NemR binds (the ‘NemR recognition box) has also been identified<sup>30</sup> (Figure 8). </p>
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<img src="https://static.igem.org/mediawiki/2014/7/72/NemR_box.jpg" style="margin-right: 300px; margin-left: 300px;"  alt="NemR box">
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<p align="center"><i><strong>Figure 8: </strong> Diagram depicting the sequence of the ‘NemR box’. Adapted from Umezawa et al.<sup>31</sup> </i></p>
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<p>&nbsp;</p>
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<p>On the assumption that TNT was able to naturally diffuse into the <i>E.coli</i> cell, we hypothesised that if we designed a promoter containing this binding site gene sequence, the promoter would therefore become inducible by the addition of TNT through the use of the NemR protein which is naturally found in <i>E.coli</i> and would be a vastly simpler TNT-detecting gene expression system as it would rely on the success of considerably fewer engineered parts.
 
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It has been shown that NemR also responds to cysteine-modifying electrophiles, including NEM, showdomycin, and, more weakly, iodoacetamide <sup>33</sup> </a>. The repressor function of NemR was inactivated through the addition of Cysteine modifying reagents. Furthermore, based upon a predicted 3D structure (Figure 9), it has been proposed that NemR utilises reversible oxidation of a conserved Cys-106  residue as the signal for confirmation change and dissociation from the DNA strand thus activating gene expression<sup>34</sup>.</p>
 
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<img alt="NemR 3D" height="604" src="https://static.igem.org/mediawiki/2014/2/25/NemR_3D.jpg" style="width: 320px; height: 424px; margin-right: 300px; margin-left: 300px;" width="410" />
 
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<p align="center"><i><strong>Figure 9: </strong>3D  molecular structure of the E. coli NemR monomer. Cysteine residues are in orange, conserved Cys-106 in red, and DNA binding helices in green<sup>33</sup>.</i></p>
 
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<p>&nbsp;</p>
 
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<p>Finally, it has been indicated that the NemR protein was responsive to Hypochlorous acid (HOCl), the active component of household bleach. Addition of bleach resulted in the expression of two detoxifying enzymes for bleach: glyoxalase I and NemA<sup>35</sup>.</p>
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<h1><span class="mw-headline" id="1">Our Parts</span></h1>
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<p>Thus, it is likely that our  vastly simplified promoter, while designed and refined by us to respond to TNT, would actually have a more ubiquitous role in responding to a range of important electrophiles and thus is ripe for characterisation not only by us but also for future iGEM teams.</p>
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<p>The 2014 Exeter iGEM Team has submitted eight parts to the iGEM Registry this year. Four of them are basic parts while four of them are composite parts. Each of the four basic parts is a simple protein coding or regulatory sequence; they each have a specific composite part in which they have the complementary sequences surrounding them; the enzymes have regulatory features such as an inducible promoter and terminator sequence, and the regulatory sequences have a reporter gene following them, so we can examine their expression.</p>
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<h2> <span class="mw-headline" id="TheNemRpromoterconstruct(BBa_K1398007)">The NemR promoter construct (<a href="http://parts.igem.org/Part:BBa_K1398007">BBa_K1398007</a>)</span></h2>
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<p> When one of our parts is said to be codon optimised for <i>E. coli</i> we mean that the genes encoding the protein from the original organism were reverse-translated and codon-optimized for expression in <i>E. coli</i> using DNA2.0 GeneGPS Technology, and synthesized as BioBrick RFC10-compatible parts and cloned into an expression optimized Vector. The constructs were transformed into the iGEM vector pSB1C3 for submission to iGEM HQ. This service was provided by <a href="https://www.dna20.com/services/genegps">DNA2.0 Inc.</a>
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<p>The development of the NemR promoter was originally as part of the design of a kill switch we intended to implement. However, it was clear that impressive kill switches have already been widely developed and used. We decided instead to focus on a NemR switch that could be used to regulate them such that the bacterium would die in the absence of TNT. When we designed the NemR promoter, we debated where the ‘NemR box’ would be inserted into the construct sequence to enable successful gene repression and expression. We eventually opted for the NemR box to be placed in-between the -12 and -33 region of the promoter as this region appeared reasonably conserved across the Anderson promoter group available to us on the registry:  <a href="http://parts.igem.org/Promoters/Catalog/Anderson"> http://parts.igem.org/Promoters/Catalog/Anderson</a>.
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<h2><span class="mw-headline" id="2">Basic Parts</span></h2>
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Thus we designed the NemR construct, <a href="http://parts.igem.org/Part:BBa_K1398007">BBa_K1398007</a> as follows:</p>
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<img src="https://static.igem.org/mediawiki/2014/3/32/NemR_construct.jpg" style="width: 277px; height: 81px; margin-right: 300px; margin-left: 300px;" alt="NemR 3D">
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398000">BBa_K1398000</a> : XenB (Xenobiotic Reductase B)</b></p>
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<p>Xenobiotic Reductase B, created for use as a Trinitrotoluene and Nitroglycerine degrading protein.</p>
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<p>A monomeric flavin that can reduce certain nitro- groups to nitrate-. Increases the resistance of organisms to the toxic effects of nitrocompounds.</p>
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<p>This sequence encodes for a protein (with an attached His (x6) Tag to allow for purification) and nothing else. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimized for expression in <I>E. coli</I>.</p>
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<p align="center"><i><strong>Figure 10: </strong> The BBa NemR construct we designed </i></p>
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398002">BBa_K1398002</a> : NemA (N-ethylmaleimide reductase)</b></p>
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<p>N-ethylmaleimide reductase, created for use as a Trinitrotoluene and Nitroglycerine degrading protein. NemA is a flavoprotein that primarily catalyses the reduction of N-ethylmaleimide (NEM), which is toxic to cell growth.</p>
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<p>However, it is also involved in the degradation of other toxic compounds for their reuse in nitrogen metabolism. Some of these compounds include PETN, quinones and chromate. It increases the resistance of organisms to the toxic effects of these compounds.</p>
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<p>This sequence only encodes for the protein, an attached His (x6) Tag to allow for purification and a double-STOP codon. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimised for expression in <I>E. coli</I>.</p>
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<p>&nbsp;</p>
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398005">BBa_K1398005</a> : NemR Upstream Intergenic Region</b></p>
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<p>This sequence is found upstream of several NemR regulated genes. It was created for use as a TNT-detection mechanism.</p>
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<p>It contains the bases from X+ to the start codon, which include a promoter and RBS. This region should allow regulation of gene products through the detection of TNT. If it is not present NemR will bind to a specified sequence of bases and inhibit transcription. If TNT is not present NemR will not bind and transcription will complete.</p>
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<p>The construct begins with the synthetic promoter NemR, which combines a high-expression promoter with the NemR recognition box (<a href="http://parts.igem.org/Part:BBa_K1398008">BBa_K1398008</a>). It is followed by a strong RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>), the fluorescent reporter iLOV (<a href="http://parts.igem.org/Part:BBa_K660004">BBa_K660004</a>), a double STOP codon and a double terminator made up of <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>.</p>
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398008">BBa_K1398008</a> : NemR Recognition Promoter</b></p>
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<p>The addition of the iLOV reporter gene was also to help further characterise part <a href="http://parts.igem.org/Part:BBa_K660004">BBa_K660004</a> submitted into the iGEM database in 2011 by the team from Glasgow: <a href="https://2011.igem.org/Team:Glasgow/LOV2"> https://2011.igem.org/Team:Glasgow/LOV2</a>. Given that this promoter would respond to TNT, this means we are also generating a biosensor for TNT.</p>
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<p>This sequences combines a high level constitutive promoter with the NemR binding box, which allows NemR to bind to DNA when no TNT is present in the cell. It was created for use as a TNT-detection mechanism. It combines <a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> with the NemR box to create a promoter that will theoretically have high levels of transcription when TNT is not present.</p>
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<p>While hybrid promoters have been designed before upon similar ideas, this is usually done to develop a switch with additional functionality to respond to the insertion of an additional engineered system (eg. For example, it is common that we utilise dual Lac and IPTG inducible promoters in the design of gene circuits).  Our promoter construct’s incredibly simple design compared to previous teams proposes the idea that by identifying the binding site of a repressor protein, anyone could theoretically make a promoter of any level of expression strength that is responsive to any chemical stimulus found in nature, and likely many that are not. </p>
 
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<p>We aim to demonstrate this future possibility through the creation of our TNT-responsive NemR-sensitive promoter. Any strong output could therefore be inhibited or, alternatively, a weakly expressed gene could have its rate of expression enhanced by identifying the correct gene sequence. Alternatively, any construct could theoretically could be made to respond to a very specific chemical signal, such as TNT. If we are able to prove that our TNT-specific promoter is successful, it will provide the proof of concept to apply this theory to millions of other chemical signals which gene circuits could be designed to be receptive to. </p>
 
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<h2><span class="mw-headline" id="3">Composite Parts</span></h2>
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398001">BBa_K1398001</a> : XenB (Inducible Construct)</b></p>
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<h2> <span class="mw-headline" id="References">References:</span></h2>
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<img src="https://static.igem.org/mediawiki/parts/2/2a/XenB_composite.png">
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<p>A construct created to degrade TNT and nitroglycerin. The construct contains the coding sequence for XenB (<a href="http://parts.igem.org/Part:BBa_1398000">BBa_1398000</a>), an enzyme with the capability to degrade nitro- groups in chemicals.</p>
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<p>The construct also contains a Lactose inducible promoter (<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a>), a strong RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_R0034</a>) and a double terminator of <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>. The protein has been codon-optimised for expression in <I>E. coli</I>.</p>
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398003">BBa_K1398003</a> : NemA (Inducible Construct)</b></p>
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<img src="https://static.igem.org/mediawiki/parts/2/2c/NemA_composite.png">
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<p>A construct created to degraded TNT and nitroglycerin. The construct contains the coding sequence for NemA (<a href="http://parts.igem.org/Part:BBa_1398002">BBa_1398002</a>), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism.</p> <p>The construct also contains a Lactose-inducible promoter (<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a>), a strong RBS (<a href="http://parts.igem.org/Part:BBa_R0034">BBa_R0034</a>) and a double terminator made up of <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>. The protein has been codon-optimised for expression in <I>E. coli</I>. </p>
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<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/9013822/">http://www.ncbi.nlm.nih.gov/pubmed/9013822/</a></li>
 
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/</a></li>
 
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/</a></li>
 
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/figure/f2/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/figure/f2/</a></li>
 
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519536/</a></li>
 
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<li><a href="http://www.jbc.org/content/288/19/13789/F3.expansion.html">http://www.jbc.org/content/288/19/13789/F3.expansion.html</a></li>
 
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<li><a href="http://www.jbc.org/content/288/19/13789.full">http://www.jbc.org/content/288/19/13789.full</a></li>
 
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<li><a href="http://www.jbc.org/content/288/19/13789.full">http://www.jbc.org/content/288/19/13789.full</a></li>
 
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398004">BBa_K1398004</a> : NemR Intergenic Reporter</b></p>
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<img src="https://static.igem.org/mediawiki/parts/8/8b/NemR_UIR_composite_new.png">
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<p>A construct created to test the effectiveness of the NemR upstream intergenic region (<a href="http://parts.igem.org/Part:BBa_1398005">BBa_1398005</a>).</p>
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<p>The construct contains the entire NemR upstream region (<a href="http://parts.igem.org/Part:BBa_1398005">BBa_1398005</a>), which contains a promoter and RBS. It is followed by the fluorescent reporter iLOV (listed in the repository as <a href="http://parts.igem.org/Part:BBa_K660004">BBa_K660004</a>), a double STOP codon and a double terminator made up of <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>.</p>
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<p>See <a href="https://2014.igem.org/Team:Exeter/Detection#Results">Detection of Xenobiotics</a> for the results of testing.</p>
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<p><b><a href="http://parts.igem.org/Part:BBa_K1398007">BBa_K1398007</a> : NemR Promoter Reporter</b></p>
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<img src="https://static.igem.org/mediawiki/parts/3/36/NemR_P_composite.png">
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<p>A construct created to test the effectiveness of the NemR recognizing promoter (BBa_1398008).</p>
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<p>The construct begins with the synthetic promoter NemR, which combines a high-expression promoter (<a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>) with the NemR recognition box (<a href="http://parts.igem.org/Part:BBa_K1398008">BBa_K1398008</a>). It is followed by a strong RBS (<a href="http://parts.igem.org/Part:BBa_R0034">BBa_R0034</a>), the fluorescent reporter iLOV (<a href="http://parts.igem.org/Part:BBa_K660004">BBa_K660004</a>), a double STOP codon and a double terminator made up of <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a></p>
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<p>See <a href="https://2014.igem.org/Team:Exeter/Detection#Results">Detection of Xenobiotics</a> for the results of testing.</p>
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<h2>Navigation</h2>
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<p><a href="https://2014.igem.org/Team:Exeter/iLOVCharacterisation">Previous: iLOV Characterisation </a></p>

Latest revision as of 22:48, 17 October 2014

Exeter | ERASE

Contents

Our Parts

The 2014 Exeter iGEM Team has submitted eight parts to the iGEM Registry this year. Four of them are basic parts while four of them are composite parts. Each of the four basic parts is a simple protein coding or regulatory sequence; they each have a specific composite part in which they have the complementary sequences surrounding them; the enzymes have regulatory features such as an inducible promoter and terminator sequence, and the regulatory sequences have a reporter gene following them, so we can examine their expression.

When one of our parts is said to be codon optimised for E. coli we mean that the genes encoding the protein from the original organism were reverse-translated and codon-optimized for expression in E. coli using DNA2.0 GeneGPS Technology, and synthesized as BioBrick RFC10-compatible parts and cloned into an expression optimized Vector. The constructs were transformed into the iGEM vector pSB1C3 for submission to iGEM HQ. This service was provided by DNA2.0 Inc.

Basic Parts

BBa_K1398000 : XenB (Xenobiotic Reductase B)

Xenobiotic Reductase B, created for use as a Trinitrotoluene and Nitroglycerine degrading protein.

A monomeric flavin that can reduce certain nitro- groups to nitrate-. Increases the resistance of organisms to the toxic effects of nitrocompounds.

This sequence encodes for a protein (with an attached His (x6) Tag to allow for purification) and nothing else. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimized for expression in E. coli.


BBa_K1398002 : NemA (N-ethylmaleimide reductase)

N-ethylmaleimide reductase, created for use as a Trinitrotoluene and Nitroglycerine degrading protein. NemA is a flavoprotein that primarily catalyses the reduction of N-ethylmaleimide (NEM), which is toxic to cell growth.

However, it is also involved in the degradation of other toxic compounds for their reuse in nitrogen metabolism. Some of these compounds include PETN, quinones and chromate. It increases the resistance of organisms to the toxic effects of these compounds.

This sequence only encodes for the protein, an attached His (x6) Tag to allow for purification and a double-STOP codon. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimised for expression in E. coli.


BBa_K1398005 : NemR Upstream Intergenic Region

This sequence is found upstream of several NemR regulated genes. It was created for use as a TNT-detection mechanism.

It contains the bases from X+ to the start codon, which include a promoter and RBS. This region should allow regulation of gene products through the detection of TNT. If it is not present NemR will bind to a specified sequence of bases and inhibit transcription. If TNT is not present NemR will not bind and transcription will complete.


BBa_K1398008 : NemR Recognition Promoter

This sequences combines a high level constitutive promoter with the NemR binding box, which allows NemR to bind to DNA when no TNT is present in the cell. It was created for use as a TNT-detection mechanism. It combines BBa_J23100 with the NemR box to create a promoter that will theoretically have high levels of transcription when TNT is not present.


Composite Parts

BBa_K1398001 : XenB (Inducible Construct)

A construct created to degrade TNT and nitroglycerin. The construct contains the coding sequence for XenB (BBa_1398000), an enzyme with the capability to degrade nitro- groups in chemicals.

The construct also contains a Lactose inducible promoter (BBa_R0010), a strong RBS (BBa_R0034) and a double terminator of BBa_B0010 and BBa_B0012. The protein has been codon-optimised for expression in E. coli.


BBa_K1398003 : NemA (Inducible Construct)

A construct created to degraded TNT and nitroglycerin. The construct contains the coding sequence for NemA (BBa_1398002), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism.

The construct also contains a Lactose-inducible promoter (BBa_R0010), a strong RBS (BBa_R0034) and a double terminator made up of BBa_B0010 and BBa_B0012. The protein has been codon-optimised for expression in E. coli.


BBa_K1398004 : NemR Intergenic Reporter

A construct created to test the effectiveness of the NemR upstream intergenic region (BBa_1398005).

The construct contains the entire NemR upstream region (BBa_1398005), which contains a promoter and RBS. It is followed by the fluorescent reporter iLOV (listed in the repository as BBa_K660004), a double STOP codon and a double terminator made up of BBa_B0010 and BBa_B0012.

See Detection of Xenobiotics for the results of testing.


BBa_K1398007 : NemR Promoter Reporter

A construct created to test the effectiveness of the NemR recognizing promoter (BBa_1398008).

The construct begins with the synthetic promoter NemR, which combines a high-expression promoter (BBa_J23100) with the NemR recognition box (BBa_K1398008). It is followed by a strong RBS (BBa_R0034), the fluorescent reporter iLOV (BBa_K660004), a double STOP codon and a double terminator made up of BBa_B0010 and BBa_B0012

See Detection of Xenobiotics for the results of testing.


Navigation

Previous: iLOV Characterisation

Exeter | ERASE

Retrieved from "http://2014.igem.org/Team:Exeter/Parts"