Team:Exeter/Project

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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
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<div id="toctitle"><h2>Contents</h2></div>
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<li class="toclevel-1"><a href="#TheSolution:OldYellowEnzymes"><span class="tocnumber">1</span> <span class="toctext">Old Yellow Enzymes</span></a>
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<li class="toclevel-1"><a href="#1"><span class="tocnumber">1.</span> <span class="toctext">Overview</span>
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<li class="toclevel-1"><a href="#XenB(BBa_K1398001)"><span class="tocnumber">2</span> <span class="toctext">XenB (BBa_K1398001)</span></a></li>
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<ul>
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<li class="toclevel-2"><a href="#DegradationofTNT"><span class="tocnumber">2.1</span> <span class="toctext">Degradation of TNT</span></a></li>
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<li class="toclevel-2"><a href="#DegradationofNitroglycerin"><span class="tocnumber">2.2</span> <span class="toctext">Degradation of Nitroglycerin</span></a>
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<li class="toclevel-2"><a href="#TheXenBConstruct:(BBa_K1398001)"><span class="tocnumber">2.3</span> <span class="toctext">The XenB Construct: (BBa_K1398001)</span></a></li>
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</ul>
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</li>
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<li class="toclevel-1"><a href="#NemA(BBa_K1398002)"><span class="tocnumber">3</span> <span class="toctext">NemA (BBa_K1398002)</span></a></li>
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<ul>
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<li class="toclevel-2"><a href="#TheNemAConstruct:(BBa_K1398002)"><span class="tocnumber">3.1</span> <span class="toctext">The NemA Construct: (BBa_K1398002)</span></a></li>
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</ul>
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<li class="toclevel-1"><a href="#References"><span class="tocnumber">4</span> <span class="toctext">References</span></a></li>
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<li class="toclevel-1"><a href="#2"><span class="tocnumber">2.</span> <span class="toctext">Background </span>
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<li class="toclevel-1"><a href="#3"><span class="tocnumber">3.</span> <span class="toctext">The Project</span>
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<h1> <span class="mw-headline" id="TheSolution:OldYellowEnzymes">Old Yellow Enzymes</span></h1>
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<ul>
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<li class="toclevel-1"><a href="#3.1"><span class="tocnumber">3.1</span> <span class="toctext">Modelling</span>
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<li class="toclevel-1"><a href="#3.2"><span class="tocnumber">3.2</span> <span class="toctext">Explosive degradation/Transformation </span>
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<li class="toclevel-1"><a href="#3.3"><span class="tocnumber">3.3</span> <span class="toctext">TNT Detection</span>
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<li class="toclevel-1"><a href="#3.4"><span class="tocnumber">3.4</span> <span class="toctext">Biosafety</span>
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<p> Several microbial enzymes have the ability to catalyse the break-down of nitroaromatic compounds such as TNT and NG. These enzymes fall into two main families: </p>
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<ol>
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<li><strong>Oxygen-Insensitive Nitroreductases:</strong> These enzymes sequentially perform two-electron reductions of nitro groups. They typically contain Flavin mononucleotides FMN which they use, along with NADPH as a cofactor and electron donor. Examples include the nitroreductases NfsA and NfsB from <i>Escherichia coli</i><sup>1</sup>, PnrA and PnrB from <i>Pseudomonas putida</i><sup>2</sup> , and NitA and NitB from <i>Clostridium acetobutylicum</i><sup>3</sup>. </li>
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<li><strong>Old Yellow Enzymes (OYE):</strong> The physiological function of this family of NADPH dehydrogenases is not yet well established; however, they are often associated with nitroaromatic compound reduction. Within the OYE family, two types of enzymes have been described:</li>
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<p>Type I hydride transferases, which, like the oxygen-insensitive nitroreductases above, reduce the nitroaromatic compounds to hydroxylamine derivatives</p>
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<p>Type II hydride transferases, which catalyse a nucleophilic attack on the aromatic ring of TNT<sup>4,5</sup></p>
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</ul>
</ul>
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<li class="toclevel-1"><a href="#4"><span class="tocnumber">4.</span> <span class="toctext">Our Parts</span>
</ul>
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<p>Of bacterial OYE family members, those that are the best characterized are XenA - XenF of <i>P. putida</i> KT24406 XenB from <i>Pseudomonas fluorescens</i><sup>7</sup>, PETN reductase from <i>Enterobacter cloacae</i> PB28, NemA reductase from <i>E. coli</i><sup>9</sup> and YqjM from <i>Bacillus subtilis</i><sup>10</sup>.</p>
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<p>In 2009 the <a href= "https://2009.igem.org/Team:Edinburgh">Edinburgh iGEM team</a> developed the concept of a Nitrate/Nitrite biosensor which could be used to detect TNT. They generated a biobrick (<a href="http://parts.igem.org/Part:BBa_K216006">BBa_K216006</a>) for the gene <i>onr</i> (organic nitrate reductase) that encodes pentaerythritol tetranitrate (PETN) reductase to function as a TNT degrader. However, neither were completely characterised. </p>
 
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<p>We sought to find new, mostly uncharacterised, enzymes to function as additional solutions to offer alternative mechanisms which may be more suited to particular problems. Given that the PETN reductase was a member of the Old Yellow Enzyme family, we decided that we would target additional members of the OYE group to help expand the both the range of enzymes that can be utilised to degrade explosives and the range of unexploded ordnance chemicals that could be degraded to those that contain nitroglycerin.</p>
 
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<p>Our initial shortlist contained the following the proteins: XenA, XenB, NemA and YqjM''' . From these we selected two to examine over the summer. These were <strong>XenB</strong> and <strong>NemA</strong> which have been submitted to the Registry of Standard Biological Parts as BBa_K1398001 and BBa_K1398002 respectively.</p>
 
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<h1> <span id="1">Overview </span></h1>
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<h1> <span class="mw-headline" id="XenB(BBa_K1398001)">XenB (<a href="http://parts.igem.org/Part:BBa_K1398001">BBa_K1398001</a>)</span></h1>
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<p>The University of Exeter’s 2014 iGEM team’s project is called:
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E.R.A.S.E.
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Explosive Remediation by Applied Synthetic E. coli.
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<p> XenB is an NADH-dependent flavoprotein (Xenobiotic Reductase B) from the soil bacterium ''Pseudomonas fluorescens''. We chose XenB because:</p>
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We aim to design a biological system that will enable safe bioremediation and detection of two of the most common explosives: <b>TNT</b> and <b>Nitroglycerin</b> (NG). As proof-of-principle we have performed this work in <i>E. coli</i>.
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<ol>
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</p>
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<li>XenB may serve a dual purpose as a Nitroglycerin and TNT degrading enzyme (see below).</lI>
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<li>The phylogeny of OYE members<sup>11</sup> suggests 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<sup>12</sup> enhancing our chances of successful expression. </li>
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</ol>
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<h2><span id="2"> Background </span></h2>
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<h2> <span class="mw-headline" id="DegradationofTNT">Degradation of TNT</span></h2>
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<p> TNT and NG are some of the most ubiquitous chemicals used in industrial and military explosives. This includes their use on demolition sites, in landmines and as other explosive remnants of war (<a href="https://www.icrc.org/en/war-and-law/weapons/explosive-remnants-war">ERW</a>). However, whilst humanitarian concerns surrounding the explosive properties of TNT and NG are likely to be the first association we have with these chemicals, they are also toxic environmental pollutants. These can be the remnants of munitions factories, as well as mining and building sites, around the world. Unused munitions are difficult to dispose of with dumping sites a common solution. Many munitions, both dumped and planted, are able to leak into the surrounding soil, which in turn causes environmental pollution. Please see<a href="https://2014.igem.org/Team:Exeter/TheProblem"> The Problem </a> for more information.</p>
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<p> Pak et al. (2000)<sup>14</sup> reported the purification of the NADH-dependent flavoprotein oxidoreductase xenobiotic reductase B (XenB) from <i>Pseudomonas fluorescens</i>. </p>
 
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<p> Those authors provide a guide to some of the degradative reactions of XenB from <i>P. fluorescens</i>. This indicates that XenB may catalysed the reduction of TNT, either by adding a hydride to the aromatic ring and forming a dihydride Meisenheimer complex<sup>15</sup> or by catalysing the reduction of nitro groups directly (Figure 1).
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<h2><span id="3">The Project</span></h2>
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However several unidentified products and proposed intermediates indicate that this process is yet to be fully elucidated.</p>
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<img alt="XenB" src="https://static.igem.org/mediawiki/2014/e/e6/Exeter_XenB.jpg" style="margin-right: 200px; margin-left: 200px;" />
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<p>We therefore sought to design a system that would:
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<p align="center"><i><strong>Figure 1:</strong> Diagram depicting the potential reductive steps catalysed by XenB. (Adapted from Pak et al. <sup>14</sup>)</i></p>
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<p>&nbsp;</p>
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<p> XenB from <i>Pseudomonas fluorescens</i> is not the only XenB enzyme that exists. A more commonly studied XenB enzyme is found in <i>Pseudomonas putida</i>. This XenB shares 88% identity to our chosen XenB from <i>P. fluorescens</i><sup>16</sup>. </p>
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<p> XenB from <i>P. putida</i> (as well as other xenobiotic reductases) demonstrate type II hydride transferase activity against TNT. Research into these xenobiotic reductases provided a greater understanding of the potential products and intermediates generated in the reactions with TNT (Figure 2).  Using figure 1 as a model,  in the mechanisms suggested by Pak et al. (2000) (above) it seems likely that the unknown m/z 196 compound was 4ADNT and that the proposed bridge product (m/z= 376) is instead linked by an amine group bridge (Figure 2). </p>
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<img height="561" src="https://static.igem.org/mediawiki/2014/5/56/Exeter_XenB_2.jpg" style="margin-right: 100px; margin-left: 100px; width: 655px; height: 395px;" width="928" />
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<p align="center"><i><strong>Figure 2:</strong> Diagram depicting the known reductive steps catalysed by XenB. (Adapted from van Dillewijn et al. <sup>17</sup>)</I></p>
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<p>&nbsp;</p>
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<p> We were unable to find more recent attempts to characterise the mechanism of XenB from <i>P. fluorescens</i>. However, given that these mechanisms for <i>P. putida</i> Xenobiotic reductase proteins are similar to the mechanisms of the type II hydride transferase family<sup>18</sup> it suggests we are more likely to find the amine bridged products in Figure 2. </p>
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<h2> <span class="mw-headline" id="DegradationofNitroglycerin">Degradation of Nitroglycerin</span></h2>
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<p> XenB also displays an apparent capacity to denitrify Nitroglycerin (NG).  It has been demonstrated that XenB catalyses the NADH-dependent cleavage of nitro groups from NG, releasing nitrite (Figure 3).
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<i>P. fluorescens</i> XenB also exhibits five-fold regioselectivity for removal of the central nitro group from NG compared to <i>P. putida</i> XenB. </p>
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<img src="https://static.igem.org/mediawiki/2014/8/82/Exeter_XenB_GTN.jpg" style="margin-right: 100px; margin-left: 100px;" alt="XenB GTN">
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<p align="center"><i><strong>Figure 3:</strong> Simplistic diagram depicting the chemistry involved in Glycerol Trinitrate transformation by XenB19.</i></p>
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<p>&nbsp;</p>
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<p>As nitrate is released from the degradation of Nitroglycerin there is a suggestion that construct could be built upon to work simultaneously as an explosive degrader and soil enricher, thus taking an environmental toxicant and converting it into an environmental benefit. </p>
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<h2> <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>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>)<sup>20</sup> and this forms the functional unit of our construct (<a href="http://parts.igem.org/Part:BBa_K1398001">BBa_K1398001</a>) (Figure 4):  </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 align="center"><i><strong> Figure 4: </strong>The XenB construct depicting the constituent parts of the construct.</i></p>
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<p>&nbsp;</p>
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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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<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> and
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<a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>.</p>
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<h1> <span class="mw-headline" id="NemA(BBa_K1398002)">NemA (<a href="http://parts.igem.org/Part:BBa_K1398002">BBa_K1398002</a>)</span></h1>
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<p> The second of our enzymes is the detoxification enzyme N-Ethylmaleimide (NEM) reductase from <i>Escherichia coli</i> encoded by the gene nemA<sup>21</sup>). Often referred to simply as NemA, the flavin-dependent NEM Reductase was chosen for several reasons: </p>
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<ol>
<ol>
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<li> Firstly, like XenB, NemA has a dual capacity to degrade both TNT and Nitroglycerin.
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<li>Degrade TNT or NG. Importantly this should be to a non-toxic product.</li>
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<li> NemA has a high degree of homology (87% identical) to pentaerythritol tetranitrate (PETN) reductase (<a href="http://parts.igem.org/Part:BBa_K216006">BBa_K216006</a>) from <i>Enterobacter cloacae</i><sup>22</sup>. The <i>E. cloacae</i> PETN reductases and <i>E. coli</i> NEM reductase showed broadly similar activity profiles, with high activity against nitrate esters<sup>23</sup>. However, NemA was shown to have a higher substrate preference for PETN and Nitroglycerin than PETN reductase, the uncharacterised gene of which was submitted by the iGEM team from Edinburgh in 2009 William’s et al. (2004)<sup>24</sup>. 
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<li>Detect TNT or NG in a sample.</li>
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<li> Finally, NemA was found to already exist in <i>E.coli</i>, albeit at a low expression level. This suggested to us a capability to be expressed successfully. However, we also realised this meant that we would more likely to face natural control mechanisms that could silence the activity of the enzyme if it is overexpressed. It also meant that we would need to assess the natural tolerance and expression levels of our <i>E.coli</i> DH5α strain to the explosives to ensure our engineered enzyme provided enhanced tolerance to TNT and NG compared to the wild-type. 
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<li>Terminate all cellular viability when there was no TNT or NG remaining in the sample.</li>  
</ol>
</ol>
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<p> Through a comparative study of PETN, NemA, Morphinone reductase and the yeast OYE, the TNT mechanism for the similar PETN reductase was detailed. Due to the sequence similarity, we are using this as a model for the pathways involved in anaerobic degradation of TNT by NemA (Figure 5). </p>
 
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<img alt="PETN reductase" src="https://static.igem.org/mediawiki/2014/3/30/Exeter_PETN_reductase_steps.jpg" style="margin-right: 250px; margin-left: 250px;" />
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<h3><span id="3.1">Modelling</span> </h3>
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<p> The first page <a href="https://2014.igem.org/Team:Exeter/Modelling">Modelling</a> details the design and modelling of our proposed system, and the influence this had on our choice of experiments.
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<p align="center"><i><strong>Figure 5: </strong>Diagram depicting the potential mechanistic steps in the transformation of TNT by PETN reductase. (Adapted from Rylott et al. <sup>25</sup> and Williams et al. <sup>26</sup>)</i></p>
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<h3><span id="3.2">Explosive degradation/Transformation</span> </h3>  
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<p>&nbsp;</p>
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<p>In the degradation/transformation aspect of the project, we are focussed the development of two parts containing the bacterial enzymes, NemA and XenB. While there have been projects done utilising different bacterial enzymes <a href="http://www.ncbi.nlm.nih.gov/pubmed/10331811/">2</a><a href="http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html">3</a> what is different about our project is that these enzymes have not been adapted for this purpose before and no previous projects have also looked at Nitroglycerin. These enzymes that we’ve selected have been shown to have the capacity to transform the toxic chemicals TNT and Nitroglycerin into non-explosive, non-toxic products.<p>See <a href="https://2014.igem.org/Team:Exeter/DegradationConstructs"> The Enzymes </a> for more background information on NemA and XenB.</p>
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<p> In addition to degradation of TNT and NG, NemA may also function as an efficient chromate reductase to remediate hexavalent chromium, a serious and widespread environmental pollutant<sup>27</sup>, and has been suggested to have a role in bleach detoxification. </p>
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We provide proof of concept of our explosive remediating enzymes through their characterisation both <i>in-vitro</i> (<a href="https://2014.igem.org/Team:Exeter/EnzymeValidation">Enzyme Kinetics</a> and <a href="enzyme-kinetics">HPLC</a>) and <i>in-vivo</i> (<a href="https://2014.igem.org/Team:Exeter/invivoactivity"><i> in-vivo</i>: Raman</a> and <a href="https://2014.igem.org/Team:Exeter/invivo"><i> in-vivo</i>: Observations</a>) </p>
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<h2> <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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<h3><span id="3.3">TNT Detection</span> </h3>
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<p> In order to asses whether any reporter or enzyme part we made had a basis for comparison, we investigated the <a href="https://2014.igem.org/Team:Exeter/EColiStressTesting"> Xenobiotic Tolerance </a> of our <i>E.coli</i> strains to gauge toxicity levels to TNT and NG. </p>
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<p>We have designed a new and simple biosensor that could  be used to detect TNT  in the form of a modified hybrid promoter which functions like a TNT detection switch. We would aim to use this to turn expression of a reporter gene on or off depending on the presence of TNT in the environment.
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<p>See <a href="https://2014.igem.org/Team:Exeter/Detection"> Detection of Xenobiotics </a> for more information on the NemR promoter constructs. </p>
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<p>In order to create what we think would be the most effective biosensor we have also chosen to further characterise the reporter iLOV which has numerous benefits over the commonly used GFP in many situations <a href="http://www.pnas.org/content/105/50/20038.full">4</a>)  . This was produced as a part on the iGEM database (<a href="http://parts.igem.org/Part:BBa_K660004">BBa_K6600004</a>) by the <a href= "https://2011.igem.org/Team:Glasgow">Glasgow 2011</a> iGEM team. </p>
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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 6): </p>
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<p>See <a href="https://2014.igem.org/Team:Exeter/iLOVCharacterisation">iLOV Characterisation</a> for more information and our characterisation of the iLOV reporter.</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 id="3.4">Biosafety </span></h2>
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<p align="center"><i><strong>Figure 6: </strong> The NemA construct depicting the constituent parts of the construct. </i></p>
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<p>We also thought about the use of this NemR promoter in the development of a kill-switch to prevent gene flow if we were to release our organism into the environment. Ideally, this would involve the organism of choice failing to produce an antidote to constitutively expressed fatal chemicals once the source of TNT has been extinguished.</p>
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<p>&nbsp;</p>
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<p>See <a href="https://2014.igem.org/Team:Exeter/Kill_Switches">Kill Switches</a> for some of our experiments testing kill switch ideas.</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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<h2><span id="4">Our Parts</span></h2>
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<p> Finally, to see the biobricks that make up the parts, see <a href="https://2014.igem.org/Team:Exeter/Parts">Our Parts</a>.</p>
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<h2> Navigation </h2>
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<p><a href="https://2014.igem.org/Team:Exeter/Modelling">Next: Modelling </a></p>
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<h1> <span class="mw-headline" id="References">References:</span></h1>
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<ol>
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<li>Zenno et al., 1996 NfsA</li>
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<li>Caballero et al., 2005 PnrA/B</li>
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<li>Kutty and Bennett, 2005 NitA/B</li>
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC92374">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC92374</a></li>
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576699/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576699/</a></li>
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576699/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576699/</a></li>
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC92374">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC92374</a></li>
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC427764">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC427764</a></li>
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<li>Miura et al.1997 NemA</li>
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<li>YqjM from Bacillus subtilis (Fitzpatrick et al.,2003).</li>
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<li><a http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576699/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576699/</a></li>
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<li><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC103757/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC103757/</a></li>
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Latest revision as of 01:46, 18 October 2014

Exeter | ERASE

Contents

Overview

The University of Exeter’s 2014 iGEM team’s project is called: E.R.A.S.E. Explosive Remediation by Applied Synthetic E. coli. We aim to design a biological system that will enable safe bioremediation and detection of two of the most common explosives: TNT and Nitroglycerin (NG). As proof-of-principle we have performed this work in E. coli.

Background

TNT and NG are some of the most ubiquitous chemicals used in industrial and military explosives. This includes their use on demolition sites, in landmines and as other explosive remnants of war (ERW). However, whilst humanitarian concerns surrounding the explosive properties of TNT and NG are likely to be the first association we have with these chemicals, they are also toxic environmental pollutants. These can be the remnants of munitions factories, as well as mining and building sites, around the world. Unused munitions are difficult to dispose of with dumping sites a common solution. Many munitions, both dumped and planted, are able to leak into the surrounding soil, which in turn causes environmental pollution. Please see The Problem for more information.

The Project

We therefore sought to design a system that would:

  1. Degrade TNT or NG. Importantly this should be to a non-toxic product.
  2. Detect TNT or NG in a sample.
  3. Terminate all cellular viability when there was no TNT or NG remaining in the sample.

Modelling

The first page Modelling details the design and modelling of our proposed system, and the influence this had on our choice of experiments.

Explosive degradation/Transformation

In the degradation/transformation aspect of the project, we are focussed the development of two parts containing the bacterial enzymes, NemA and XenB. While there have been projects done utilising different bacterial enzymes 23 what is different about our project is that these enzymes have not been adapted for this purpose before and no previous projects have also looked at Nitroglycerin. These enzymes that we’ve selected have been shown to have the capacity to transform the toxic chemicals TNT and Nitroglycerin into non-explosive, non-toxic products.

See The Enzymes for more background information on NemA and XenB.

We provide proof of concept of our explosive remediating enzymes through their characterisation both in-vitro (Enzyme Kinetics and HPLC) and in-vivo ( in-vivo: Raman and in-vivo: Observations)

TNT Detection

In order to asses whether any reporter or enzyme part we made had a basis for comparison, we investigated the Xenobiotic Tolerance of our E.coli strains to gauge toxicity levels to TNT and NG.

We have designed a new and simple biosensor that could be used to detect TNT in the form of a modified hybrid promoter which functions like a TNT detection switch. We would aim to use this to turn expression of a reporter gene on or off depending on the presence of TNT in the environment.

See Detection of Xenobiotics for more information on the NemR promoter constructs.

In order to create what we think would be the most effective biosensor we have also chosen to further characterise the reporter iLOV which has numerous benefits over the commonly used GFP in many situations 4) . This was produced as a part on the iGEM database (BBa_K6600004) by the Glasgow 2011 iGEM team.

See iLOV Characterisation for more information and our characterisation of the iLOV reporter.

Biosafety

We also thought about the use of this NemR promoter in the development of a kill-switch to prevent gene flow if we were to release our organism into the environment. Ideally, this would involve the organism of choice failing to produce an antidote to constitutively expressed fatal chemicals once the source of TNT has been extinguished.

See Kill Switches for some of our experiments testing kill switch ideas.

Our Parts

Finally, to see the biobricks that make up the parts, see Our Parts.

Navigation

Next: Modelling

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