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Team:Exeter/Project
From 2014.igem.org
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Overview
We are Team E.R.A.S.E, the University of Exeter’s 2014 iGEM team.
E.R.A.S.E stands for Explosive Remediation by Applied Synthetic E. coli and that is our mission statement. We aim to design new genetic parts to enable an E. coli bacterium to securely detect and safely break down two of the most common explosives: TNT and Nitroglycerin .
Background
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 60,388 recorded casualties of which 14,569 people were killed and another 43,069 were injured. While there appears to be a decline in casualties caused by mines and ERW, there were still over 2,000 casualties in 2013 with 83% of casualties being civilian. Of those civilian casualties approximately 50% were children 1 . Thus, we felt it was imperative to try and generate a safe, cost-effective method of landmine/ERW detection 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.
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.
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. 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.
See The Problem for more information.
The Project
Thus our project has two principal focuses which can be applied to both the humanitarian and environmental problems: Explosive degradation/Transformation and TNT Detection.
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. We aim to provide proof of concept of our explosive remediating enzymes through their characterization both in-vitro and in-vivo in E. coli, hence E.R.A.S.E.
See The Enzymes for more background information on NemA and XenB.
TNT 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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