First Name:
Kaitlin
Last Name:
Rzasa
Major Department:
Chemistry
Thesis Director:
Libby Puckett
Date of Thesis:
May 2013
The problem of resistance to β-lactam antibiotics has become widespread. When the antibiotics were first developed, most strains of bacteria were susceptible, but, due to over- prescription, many strains today have developed resistance. The aim of this project is to create sensing systems to monitor the hydrolysis of β-lactam antibiotics. These systems will utilize β-lactamase, a gene responsible for antibiotic resistance, and enhanced green fluorescent protein (EGFP), a pH sensitive fluorescent protein. β-Lactamase is a protein that bacteria have developed to catalyze the cleavage of the β-lactam ring present in β-lactam antibiotics, thus rendering the antibiotic inactive. The β-lactam ring is the feature that makes the antibiotic effective by preventing bacterial cell wall synthesis during replication. The inactivation of the β-lactam ring is accompanied by the release of a proton, thereby lowering the local pH. In order to monitor this catalysis, a pH dependent reporter protein is necessary. For this reason, EGFP, a variant of green fluorescent protein containing two major mutations near and within the chromophore region, was chosen. These mutations enable EGFP to respond to pH changes. In the assay, the EGFP domain of the fusion protein recognizes the drop in pH due to β-lactam hydrolysis, and its level of fluorescence decreases over time. This information can then be used to perform kinetic experiments and inhibitor studies. This project has three major directions. One, the development of an in vitro protein-based assay, which has been completed previously, but is being redone for the sake of comparison. Two, the validation of the pH theory, in which the β-lactamase and EGFP proteins are expressed separately; and three, the development of an in vivowhole-cell based system to measure the bioavailability of new β-lactam antibiotics. Both the in vitro and in vivosystems will be useful in exploring new β-lactamase inhibitors as well as new antibiotic cocktails. The 3 individual proteins will be used for side-by-side comparisons with the previously developed in vitro assay.