Optimization Of Parameters For The Development Of Sensing Systems To Measure Hydrolysis Mechanisms With Enhanced Green Fluorescent Protein

The purpose of this research is to develop sensing systems capable of measuring two hydrolysis mechanisms using the pH-dependent fluorescent reporter protein, enhanced green fluorescent protein (EGFP). The enzyme, ß-lactamase, catalyzes the hydrolysis of ß-lactam antibiotics, notably penicillins, and releases one proton. The hydrolysis of organophosphates, catalyzed by organophosphorus hydrolase (OPH), releases two protons. The release of these protons, in each case, changes the pH of the surrounding environment. The basis for developing sensing systems with these enzymes comes from previous work, in which a fusion protein between EGFP and ß-lactamase was developed. This research showed that the pH change from the release of protons through hydrolysis is a local change, and EGFP must be in close proximity to the enzyme domain to detect these changes. To validate this theory, the local pH theory, the goal was to develop separate ß-lactamase and EGFP proteins. Additionally, creating a fusion construct with OPH and EGFP to assess the feasibility of other fusion protein systems with EGFP was attempted. The transformation of the separate ß-lactamase and EGFP plasmids proved unsuccessful. The two sets of primers designed for the OPH-EGFP experiment were successful in amplifying the EGFP gene, but only one set was sufficient in amplifying the OPH gene. Further optimization of PCR parameters is needed for both experiments to increase the DNA concentration in the samples and ensure successful transformation into DH5a cells. With the threat of the toxic effects of organophosphates, which are found in pesticides and chemical warfare agents, and the growing problem of bacterial resistance to antibiotics, this research has far-reaching implications in the areas of agriculture and medicine. Ultimately, whole cell sensing systems will be developed to assess enzyme kinetics and perform bioavailability and toxicity studies.