Project Details
Description
Whole cell biosensor constructs are intrinsically modular lending themselves to a synthetic biology approach. Our proposed research involves screening mutated expression libraries of the genetic regulatory element XylR to identify recognition elements displaying high binding affinity and specificity towards TNT, then use optical waveguide sensing to develop a proof-of principle whole-cell-biosensor for the recognition of TNT. To achieve this XylR will we will create populations of mutated xylR sequences using methods of directed evolution and use fluorescence-activated, cell sorting techniques to identify XylR variants with improved TNT binding. We propose to use secreted alkaline phosphatase (SEAP) as the reporter, followed by an amplification stage where the SEAP converts non-detectable small molecule substrates into a detectable form. The substrates we will test are fluorescein diphosphate and p-nitrophenyl phosphate.
We have developed a new concept in optical waveguide sensing, the leaky waveguide sensor which we will use to detect the TNT induced fluorescent products. In these devices, the waveguide layer, is a low index material (which can be a gel or involatile liquid) instead of a high-index, solid material. When the product molecules diffuse into the gel they can be detected by their fluorescence. Because the waveguide has a lower refractive index than the substrate but a higher refractive index than the sample layer, light is confined by Fresnel reflection at the waveguide/substrate interface and by total internal reflection at the waveguide/sample interface. The optical mode propagates through this low index medium. This provides the maximum overlap between the light and the chemistry, thus maximising sensitivity to changes in fluorescence caused by SEAP in the waveguide layer. These devices are especially efficient at exciting fluorescence, as the light intensity in the waveguide is higher than the external intensity.
We have developed a new concept in optical waveguide sensing, the leaky waveguide sensor which we will use to detect the TNT induced fluorescent products. In these devices, the waveguide layer, is a low index material (which can be a gel or involatile liquid) instead of a high-index, solid material. When the product molecules diffuse into the gel they can be detected by their fluorescence. Because the waveguide has a lower refractive index than the substrate but a higher refractive index than the sample layer, light is confined by Fresnel reflection at the waveguide/substrate interface and by total internal reflection at the waveguide/sample interface. The optical mode propagates through this low index medium. This provides the maximum overlap between the light and the chemistry, thus maximising sensitivity to changes in fluorescence caused by SEAP in the waveguide layer. These devices are especially efficient at exciting fluorescence, as the light intensity in the waveguide is higher than the external intensity.
Layman's description
The proliferation of explosives, new technologies, and expertise increases the potential for terrorists to evade our existing countermeasures at points of entry to and exit from the UK. Present methods for the detection of explosives leave much to be desired. To allow analysis to occur in the 'field' there is an urgent need for the development of on-site testing. There is also a need to detect explosives pollution on military sites. Toxic explosives residues are released into the environment on military training ranges as a result of incomplete detonation of munitions. As ecplosives are toxic it is therefore essential that the MoD consider the fate and transport mechanisms and the subsequent impact of these materials on the terrestrial environment. The availability of benchmark data regarding the transport and fate of the energetic materials will support predictions and modeling to enable the MoD to use best management practices for the stewardship of military sites. The results obtained will provide potential cost savings, since such knowledge will mitigate contamination and alleviate the need for future clean-up operations.
To address these challenge, we are aiming to develop novel inexpensive sensors to rapidly and effectively detect explosives. We intend to construct a proof-of-principle cell-based sensor where the cells specifically respond to the explosive TNT and produce a signal that is detected by an optical device. This requires engineering a genetic regulatory protein that specifically binds TNT. The TNT bound regulatory complex then binds to the promoter region of a reporter gene resulting in the expression of an enzyme alkaline phosphatase which is secreted out of the microbial cell. The secreted enzyme then activates a fluorescent compound and this signal is then detected using an optical waveguide sensor. The development of a genetic regulatory element that specifically recognises explosives would result in a step-change towards the development of a biosensor with strong relevance to the security and defence area.
To address these challenge, we are aiming to develop novel inexpensive sensors to rapidly and effectively detect explosives. We intend to construct a proof-of-principle cell-based sensor where the cells specifically respond to the explosive TNT and produce a signal that is detected by an optical device. This requires engineering a genetic regulatory protein that specifically binds TNT. The TNT bound regulatory complex then binds to the promoter region of a reporter gene resulting in the expression of an enzyme alkaline phosphatase which is secreted out of the microbial cell. The secreted enzyme then activates a fluorescent compound and this signal is then detected using an optical waveguide sensor. The development of a genetic regulatory element that specifically recognises explosives would result in a step-change towards the development of a biosensor with strong relevance to the security and defence area.
| Status | Finished |
|---|---|
| Effective start/end date | 31/12/12 → 31/10/14 |
Funding
- BBSRC: £74,484.37