Team:Exeter/Parts

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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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<li class="toclevel-1"><a href="#TheNemRpromoterconstruct"><span class="tocnumber">1.1</span> <span class="toctext">The NemR promoter constructs</span></a></li>
 
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<h1> <span class="mw-headline" id="TheXenBConstruct:(BBa_K1398001)">The XenB Construct: (<a href="http://parts.igem.org/Part:BBa_K1398001">BBa_K1398001</a>)</span></h2>
 
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<p> XenB is an NADH-dependent flavoprotein (Xenobiotic Reductase B) from the soil bacterium <i>Pseudomonas fluorescens</i>((<a href="http://parts.igem.org/Part:BBa_K1398001">BBa_K1398001</a>)). We chose XenB because:</p>
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<h1><span class="mw-headline" id="1">Our Parts</span></h1>
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<ol>
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<li>XenB may serve a dual purpose as a Nitroglycerin and TNT degrading enzyme (see (<a href="https://2014.igem.org/Team:Exeter/DegradationConstructs#TheXenBConstruct:%28BBa_K1398001%29)"> The Enzymes: XenB</a>) .</lI>
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<li>The phylogeny of Old Yellow Enzyme memberssuggests that XenB is similar in sequence to the previously submitted PETN reductase and is comparatively well understood.</li>
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<li>Heterologous expression of XenB from <i>P. fluorescens</i> has been previously demonstrated in <i>E. coli</i> DH5α enhancing our chances of successful expression. </li>
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<p>The coding sequence for <i>P. fluorescens</i> XenB has been deposited within GenBank (accession no. <a href="http://www.ncbi.nlm.nih.gov/nuccore/AF154062">AF154062</a>)and this forms the functional unit of our construct (<a href="http://parts.igem.org/Part:BBa_K1398001">BBa_K1398001</a>) (Figure 1):  </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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<img src="https://static.igem.org/mediawiki/2014/8/88/Exeter_Xen_B_construct.jpg" style="width: 258px; height: 83px;margin-right: 300px; margin-left: 300px;"  alt="XenB construct">
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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 align="center"><i><strong> Figure 1: </strong>The XenB construct depicting the constituent parts of the construct.</i></p>
 
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<p>&nbsp;</p>
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<h2><span class="mw-headline" id="2">Basic Parts</span></h2>
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<p>The construct contains the coding sequence for XenB (<a href="http://parts.igem.org/Part:BBa_K1398000">BBa_K1398000</a>). Expression of XenB is driven by a Lactose-inducible promoter (<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a>) coupled with a strong RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>). The construct is terminated using a double terminator made up of
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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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<br>
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<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and
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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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<a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>.</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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<br>
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<h1> <span class="mw-headline" id="TheNemAConstruct:(BBa_K1398003)">The NemA Construct: (<a href="http://parts.igem.org/Part:BBa_K1398003">BBa_K1398003</a>)</span></h2>
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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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<br>
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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>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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<br>
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<p> With the nemA DNA sequence identified<sup>28</sup> we were able to design our construct to evaluate NemA as follows (Figure 2): </p>
 
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<img alt="NemA construct" src="https://static.igem.org/mediawiki/2014/a/a1/Exeter_NemA_construct.jpg" style="width: 277px; height: 81px; margin-right: 300px; margin-left: 300px;" />
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<h2><span class="mw-headline" id="3">Composite Parts</span></h2>
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<p align="center"><i><strong>Figure 2: </strong> The NemA construct depicting the constituent parts of the construct. </i></p>
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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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<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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<br>
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<p>&nbsp;</p>
 
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<p> The construct contains the coding sequence for NemA (<a href="http://parts.igem.org/Part:BBa_K1398002">BBa_K1398002</a>), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism. 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_B0034</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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<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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<h1> <span class="mw-headline" id="TheNemRpromoterconstructs">The NemR promoter constructs</span></h1>
 
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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>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 two constructs, one with the entire NemR region was placed upstream of the promoter (The intergenic reporter <a href="http://parts.igem.org/Part:BBa_K1398004">BBa_K1398004</a>) and one where the NemR box was placed in-between the -12 and -33 region of the promoter (<a href="http://parts.igem.org/Part:BBa_K1398007">BBa_K1398007</a> 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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<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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<br>
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<h2> <span class="mw-headline" id="TheNemRpromoterconstruct(BBa_K1398004)">The NemR intergenic reporter construct(<a href="http://parts.igem.org/Part:BBa_K1398004">BBa_K1398004</a>)</span></h2>
 
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<p>We designed the NemR upstream intergenic construct, <a href="http://parts.igem.org/Part:BBa_K1398004">BBa_K1398004</a>, as follows:</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 align="center"><i><strong>Figure 3: </strong> The NemR intergenic construct depicting the constituent parts of the construct. </i></p>
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This construct was created to test the effectiveness of the NemR upstream intergenic region (BBa_1398005). The construct contains the entire NemR upstream region (<a href="http://parts.igem.org/Part:BBa_K1398005">BBa_K1398005</a>), which contains a promoter and RBS. It is followed by 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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<h2> <span class="mw-headline" id="TheNemRpromoterconstruct(BBa_K1398007)">The NemR box promoter construct(<a href="http://parts.igem.org/Part:BBa_K1398007">BBa_K1398007</a>)</span></h2>
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<p>We designed the NemR box insert 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 align="center"><i><strong>Figure 4: </strong> The NemR box construct depicting the constituent parts of the construct. </i></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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<br>
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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>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 also aim to generate a biosensor for TNT.</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"