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  • Modelling
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  • Enzyme Validation
  • in vivo: Raman
  • in vivo: Observations
  • HPLC
  • Kill Switches
  • Xenobiotic Tolerance
  • Detection of Xenobiotics
  • iLOV Characterisation
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Contents 1. Overview 2. Background 3. The Project 3.1 Modelling 3.2 Explosive degradation/Transformation 3.3 TNT Detection 3.4 Biosafety 4. Our Parts

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.

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