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Title: Development of an oxidative stress-responsive biosensor
Author: Howbrook, David
ISNI:       0000 0001 3582 6206
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2000
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The promoter region of the katG gene of Escherichia coli has been fused to two reporter genes GFPuv, encoding green fluorescent protein derived from Aequorea victoria and luxCDABE, encoding bacterial luciferase, from Photorhadbus luminescens to compare the qualities of these two reporters in microbial biosensor applications. In Escherichia coli both reporter systems produce stable signals. The lux construct was more sensitive at lower concentrations of hydrogen peroxide and the response time was shorter when compared with GFPuv. The latter, however, was better able to sense oxidative stress at concentrations that impaired signal output in the E. coli lux system. Low level non-induced bioluminescence was observed using the P. luminescens reporter system and this was utilised to measure EC50. As many compounds produce an increase in luminescence when incubated with this system, there is no means of specifically identifying any oxidative pollutants in the unknown sample. The system is limited to compounds that produce oxidative stress. Here we describe a system to add specificity to the stress-response whole-cell biosensor using glucose oxidase, which produces from glucose, hydrogen peroxide and gluconate. On incubation of these two adjuncts, glucose and glucose oxidase, with the pkatGlux whole cell biosensor, we found that the system was specific for glucose and had a range of sensitivity from 2 to 12 mM glucose. We propose that by adding glucose oxidase to the oxidative stress whole cell biosensor the specificity of the oxidative stress response can be increased, and by adding other oxidase enzymes the range of compounds that can be detected is expanded. There are enzymes of which the products of metabolism include glucose, beta-galactosidase converts lactose into glucose and galactose. The enzymes, beta-amylase and beta-amlyglucosidase digest starch to produce glucose and cellulases that act on cellulose to liberate glucose. Glucose oxidase then converts glucose to hydrogen peroxide and gluconate, the latter of which induces an increase in luminescence from the E. coli lux system. Thus it is possible to further develop the theme of adding in specificity to the stress response whole cell biosensor in the use of dual enzyme systems, where the first enzyme acts on the first substrate to yield glucose on to which glucose oxidase can metabolise, to yield hydrogen peroxide. If pkatGlux is incubated with a dual enzyme system then the number of compounds that can be biosensed can be increased and a greater specificity introduced. Samples may originate from lake, river or soil samples. These will not be 'clean'; they may contain organic debris, dirt and other bacteria that could interfere with the biosensing process. To this end lake and soil samples were spiked with substrates to see if direct sensing is possible, without the need for sample preparation. It was indicated that biosensing could take place in samples that originated from an aqueous environment. Where there were high levels of soil present, luminescence signal was quenched, which was restored on extraction of the substrate with appropriate solvent.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biosensors; Biosensing; Luminescence