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Title: Design of a ratiometric reporter to improve the dynamic range and sensitivity of a bacterial biosensor
Author: Davies, Gareth Edward
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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For applications from biosensor generation to synthetic biology, the ability to accurately and quantitatively generate input-output data from biological systems over a wide dynamic range is becoming increasingly important. This study has demonstrated that a simple approach utilising multiple promoters, recognising the same transcriptional activator but with differing affinities, linked to compatible reporter genes can achieve this goal. This simple system highlights that even for complex promoters with multiple binding sites we can use the ratio of two reporters to obtain accurate and reproducible input-output data for reporters contained on a high copy number plasmid without the need for any background subtraction or accurate determination of cell number. It has also demonstrated that the precise promoter strengths are not too crucial to the design, provided they are significantly different, as taking the ratio of either the high affinity/low affinity or medium affinity/low affinity channels give similar improvements over any single channel alone. The modularity of this system means that it should be possible to exchange the IPTG inducible promoter and hence place the ntrC* gene under the regulation of any environmentally important promoter. It should also be possible to simply construct different strength promoters for the same transcriptional activator by standard DNA synthesis techniques, therefore allowing direct ratiometric detection of specific active transcription factors. Finally, there is considerable interest in the biosensor field in the use of immobilised cells for field based applications. In these cases, determining the exact number of viable cells present at the time of use may be problematic and obtaining high signal levels may also be essential. In this regard, the use of ratiometric reporters from constructs on high copy number plasmids could alleviate many of these potential problems and significantly simplify the required biosensor detection equipment.
Supervisor: Wadhams, George; Biggin, Phil Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Microbiology ; Biosensor ; Microbial ; bacteria ; ratiometric ; fluorescence