Abstract:
An all- fibre fl uorescence spectroscopic system called the optrode has been developed and used by the Department of Physics at the University of Auckland for fluorescence signal measurements over the last 10 years. The aim of this thesis is to optimise the optrode for bacteria fluorescence measurements in three di fferent research applications. In this study, we have used the optrode to measure the Förrster Resonance Energy Transfer (FRET) changes. We have used the optrode in conjunction with a genetically encoded fluorescent biosensor to monitor the formation of bacterial biofi lms. Recent studies have shown that the formation of bacteria bio film depend on the levels of internal messenger called cyclic dinucleotide cyclic diguanyglate (c-di-GMP). The FRET based biosensor used in this study is derived from 2 types of proteins; cyan fluorescent protein (CFP) and yellow fl uorescent protein (YFP). These two proteins are incorporated into a biosensor that binds to c-di-GMP. The c-di-GMP levels in the bacteria are dependent on the presence or absence of iron leading to changes in FRET e fficiency. In the absence of iron, c-di-GMP levels decrease and the biosensor undergoes conformational change causing less or no FRET. In the presence of iron, cdi- GMP levels increase and more FRET is visible. In this study, we have used the optrode to measure the presence (YFP fluorescence) or the absence (CFP fl uorescence) of FRET to monitor the changes in c-di-GMP levels by calculating ratios of YFP to CFP. These results are then used as indications of FRET e ffciency and state of biofi lms. In this research, we investigated the use of the optrode to monitor the eff ect of ampicillin on bacteria over various exposure times. We used commercially available dyes to stain the bacteria in a way that distinguishes the dead bacteria from the mixture of alive and dead bacteria. The two dyes used were SYTO 9 and Propidium Iodide (PI). The fluorescent emission signals of SYTO 9 and PI were analysed to identify and quantify the ratio of alive and dead bacteria. Genetically modifi ed bacteria tagged with Green Fluorescence Protein (GFP) were used to show that the optrode can be used to study the transportation of bacteria in porous media with the help of a bench-scale column setup. The protocol for the use of optrode was optimised in accordance with the column setup to provide in-situ measurements for bioremediation purposes. The results shown in this thesis indicate that the use of optrode is versatile, cost-eff ective and highly sensitive for a variety of biomedical applications.