Masters Thesis

Quantification of Aac(6')-ib Molecules Within a Cell and Determination of the Level of Amikacin Resistance Conferred

Antibiotics are losing effectiveness as multidrug resistance among bacterial pathogens is spreading. Resistance enzymes, like AAC(6’)-Ib, allow bacteria to survive and proliferate in the presence of antibiotics. Conversely, discovery of new antibiotic compounds is decreasing; alternative strategies are needed to combat the rise and spread of antibiotic resistant bacteria. Studying the resistance enzymes can provide new insights into developing alternative strategies. This project set out to quantify the number of enzyme molecules within a cell and determine the relationship between the amount of enzyme molecules and the level of resistance granted by the number of molecules. E. coli strains carrying CRISPR-dCas9 genes and pFH3, a near-wild type plasmid expressing AAC(6’)-Ib, were created. Fluorescence assays were used to measure AAC(6’)-Ib tagged with an mMaple fluorophore. Resistance levels were determined using minimum inhibitory concentration (MIC) tests with amikacin Etest strips. It was found that the relationship between fluorescence and enzyme molecule number was linear. Additionally, resistance conferred by the enzyme was proportionally dependent on the number of enzyme molecules present within the measured range.

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