Team:Exeter/Project

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<h1> Welcome </h1>
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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">Overview</span>
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<p> We are team E.R.A.S.E, the University of Exeter’s 2014 iGEM team.</p>
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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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E.R.A.S.E stands for Explosive Remediation by Applied Synthetic <i>E. coli</i> and that is our mission statement. We aim to design new genetic parts to enable an <i>E. coli</i> bacterium to securely detect and safely break down two of the most common and deadly explosives: <strong> TNT </strong> and <strong> Nitroglycerin </strong>.</p>
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<h1> Background </h1>
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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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<p> These chemicals are some of the most ubiquitous chemicals used in industrial and military explosives including demolition sites, landmines and other explosive remnants of war (ERW). Between 1999-2013 In the 31 States Parties to the Mine Ban Treaty there have been a total of <strong>60,388 recorded casualties</strong> of which <strong>14,569 people were killed</strong> and another <strong>43,069 were injured</strong>. While there appears to be a decline in casualties caused by mines and ERW, there were still <strong>over 2,000 casualties in 2013</strong> with <strong>83% of casualties being civilian</strong>. Of those civilian casualties approximately <strong>50% were children</strong> http://www.the-monitor.org/index.php/LM/Our-Research-Products/Maputo-3rd-Review-Conference/Casualty-trends-1999-2013 . Thus, we felt it was imperative to try and generate a safe, cost-effective method of landmine/ERW detection and potentially also remediation through synthetic biology that could be applied on a global scale. We felt that the use of a synthetic bacterium specialised with a reporter and explosive degrading enzymes was one way to do this.</p>
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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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<p>Secondary to this main goal, we have also been investigating the environmental issue of detection and biotransformation of these toxic environmental pollutants which are left over as waste 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.
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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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Workers in munitions factories and those living in TNT polluted environments also suffer serious health issues including a significant increase in the development of cataracts, and multiple types of cancer including leukaemia.
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As the expensive and impractical incineration of contaminated soil remains the main solution to this environmental problem, we feel that we could improve on current efforts to use bacteria as bioremediators by developing and characterising new and superior enzymes to aid in the effort.</p>
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<li class="toclevel-1"><a href="#4"><span class="tocnumber">4.</span> <span class="toctext">Our Parts</span>
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<p>See <p><a href="https://2014.igem.org/Team:Exeter/TheProblem"> The Problem </a> for more information.</p>
 
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<h1>The Project</h1>
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<h1> <span id="1">Overview </span></h1>
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<p>Thus our project has two principal focuses which can be applied to both the humanitarian and environmental problems: <strong>Explosive degradation/Transformation</strong> and <strong>TNT Detection</strong>.</p>
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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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<h2>Explosive degradation/Transformation </h2>  
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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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</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 <p><a href="http://www.ncbi.nlm.nih.gov/pubmed/10331811/">2 <p><a href="http://school.mech.uwa.edu.au/~jamest/demining/others/ornl/rsb.html">3</a></p> 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 and explosive chemicals TNT and Nitroglycerin into non-explosive, non-toxic products. We aim to provide proof of concept of our explosive remediating enzymes through their characterization both in-vitro and in-vivo in <i>E. coli</i>, hence E.R.A.S.E.</p>
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<h2><span id="2"> Background </span></h2>
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<p>See <p><a href="https://2014.igem.org/Team:Exeter/DegradationConstructs"> The Enzymes </a></p> for more background information on NemA and XenB.</p>
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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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<h2>TNT Detection </h2>
 
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<p>We have designed a new and simple biosensor that could be used to detect TNT and buried landmines in the form of a modified hybrid promoter. This responds to a specific repressor molecule called NemR to function like a TNT detection switch. We aim to use this to turn expression of a reporter gene on or off depending on the presence of TNT in the environment. We felt that this promoter could be used primarily as the switch for a biosensor. When TNT is present in an environment, for example leaked from a buried mine or old ordnance, NemR would be bound by TNT and the promoter would enable the expression of a colourful protein under UV light.</p>
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<h2><span id="3">The Project</span></h2>
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<p>See Detection of Xenobiotics for more information on the NemR promoter.</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 http://www.pnas.org/content/105/50/20038.full . 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>See <p><a href="https://2014.igem.org/Team:Exeter/iLOVCharacterisation"> iLOV Characterisation </a></p> for more information on the iLOV reporter.</p>
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<p>We therefore sought to design a system that would:
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<ol>
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<li>Degrade TNT or NG. Importantly this should be to a non-toxic product.</li>
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<li>Detect TNT or NG in a sample.</li>
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<li>Terminate all cellular viability when there was no TNT or NG remaining in the sample.</li>
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</ol>
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<h2>Biosafety </h2>
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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>We also thought about the use of this promoter in the development of a kill-switch to prevent gene flow if released into the environment, with the organism of choice failing to produce an antidote to consistently expressed fatal chemicals once the source of TNT has been extinguished.</p>
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<h3><span id="3.2">Explosive degradation/Transformation</span> </h3>  
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<p>See Kill Switches for some of our biosafety considerations.</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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<h1> References </h1>
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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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<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>
 +
<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.
 +
<p>See <a href="https://2014.igem.org/Team:Exeter/Detection"> Detection of Xenobiotics </a> for more information on the NemR promoter constructs. </p>
 +
<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>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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<h2><span id="3.4">Biosafety </span></h2>
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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>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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<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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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

Exeter | ERASE

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