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Team:Exeter/Project
From 2014.igem.org
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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. As proof-of-principle we have performed this work in E. coli. We are Team E.R.A.S.E, the University of Exeter’s 2014 iGEM team.
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:
- Detect TNT or NG in a sample.
- Degrade TNT or NG. Importantly this should be to a non-toxic product
- Terminate all cellular viability when there was no TNT or NG remaining in the sample.
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 aim to provide proof of concept of our explosive remediating enzymes through their characterization both in-vitro and in-vivoTNT Detection
We have designed a new and simple biosensor that could theoretically 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 munitions, NemR would be bound by TNT and the promoter would enable the expression of a colourful protein under UV light.
See Detection of Xenobiotics for more information on the NemR promoter.
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 on the iLOV reporter.
Biosafety
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 constitutively expressed fatal chemicals once the source of TNT has been extinguished.
See Kill Switches for some of our biosafety considerations.
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