The Development Of Sensing Systems To Measure Hydrolysis Of ß-lactam Antibiotics

ASU Author/Contributor (non-ASU co-authors, if there are any, appear on document)
Samuel Wyatt Guy (Creator)
Appalachian State University (ASU )
Web Site:
Libby Puckett

Abstract: Antibiotic resistance is a prevalent problem in modern society, with almost three million cases of antibiotic resistant infections occurring yearly in the U.S.1 When antibiotics were developed as a treatment for disease and infection, most bacteria were susceptible to their antibiotic properties. However, over time, as a result of over-prescription and misuse of the drugs, bacteria have developed resistance to the medications. ß-lactam antibiotics are a common and effective class of antibiotics, but they too are losing potency as more bacteria develop resistance to them in the form of ß-lactam hydrolysis caused by ß-lactamase. ß-lactamase is an enzyme that catalyzes the cleavage of the ß-lactam ring of penicillin antibiotics, rendering them ineffective. The goal of this project is to create sensing systems that can monitor the hydrolysis of ß- lactam antibiotics. The systems will utilize a fusion protein of ß-lactamase and enhanced green fluorescent protein (EGFP). ß-lactamase, an enzyme responsible for conferring antibiotic resistance, is encoded in the ampicillin resistance (ampr) gene of many plasmids. The ß-lactam ring of penicillins gives the antibiotics their effectiveness by inhibiting cell wall synthesis during bacterial replication. When ß-lactamase catalyzes the cleavage the ß-lactam ring, a proton is released, decreasing the local pH. EGFP, a variant of green fluorescent protein (GFP), is a pH-sensitive fluorescent protein that will be used as the reporter protein to monitor the hydrolysis of the ß-lactam ring. EGFP has two key mutations in the chromophore region that result in its fluorescence being more intense than that of GFP when excited at 488 nm and allows it to respond to changes in pH. In the assays being developed, the EGFP domain of the fusion protein will respond to the drop in local pH resulting from the ß-lactam hydrolysis, leading to a decrease in fluorescence over time.

Additional Information

Honors Project
Guy, S. (2020). The Development Of Sensing Systems To Measure Hydrolysis Of ß-lactam Antibiotics. Unpublished Honors Thesis. Appalachian State University, Boone, NC.
Language: English
Date: 2020
Sensing System, ß-lactamase, ß-lactam Antibiotics, Enhanced Green Flourescent Protein

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