UBC Theses and Dissertations
Characterization of a novel bacterial transducer based on genetically engineered bioluminescence Blouin, Kim
The research described in this thesis is aimed at helping with the development of better sensors for instrumentation involved in the monitoring of otherwise difficult-todetect chemical compounds and their toxic effects. The proposed transducer is based on genetically engineered bioluminescence in the biofilm-forming bacterium Caulobacter crescentus. We studied the biochemical mechanisms involved in the bioluminescence process which revealed a complex multi-phase kinetic behaviour. The bioluminescence profiles were characterized in terms of relevant protocol-related parameters such as substrate concentration, environment, order of mixing, etc. A coupled-enzyme model of the bioluminescence mechanism is proposed in order to explain and interpret the complex profiles. The interpretation is supported by an in vitro analysis isolating the two enzymes directly involved in in vivo bioluminescence. This study demonstrates the importance of precise control of protocol-related parameters required to obtain reproducible and meaningful results. A rudimentary toxicity protocol was developed based on the knowledge gained during the characterization studies. The results obtained were comparable to previously published data and hence demonstrated the considerable potential of the bacterial transducer for use as a toxicity sensor. The data generated also supported the validity of the model proposed during the characterization of the bioluminescence mechanism. The bacterial transducer was thus found to be appropriate for future application in a flow-through toxicity assessing instrumentation system. The first stage of this next phase of the project was accomplished by designing and building a prototype of a flow-through system. Some preliminary flow-through data were generated which demonstrated that the prototype (and the flow-through concept) are indeed advantageous and worthy of further development. Some suggestions for specific directions for such further development are outlined and discussed, as well as some other applications for the flow-through instrumentation system.
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