Mentor: John Alumasa, Ph.D.

Antibiotic resistance is a global health crisis that is expected to continue to evolve and cause millions of deaths annually. As increasing amounts of antibiotics are misused in humans, agriculture, and animals, bacteria learn to adapt and become resistant to them, making common infections and diseases harder to treat and even more deadly. Tetracycline is a common antibiotic used to treat bacterial infections through inhibiting bacterial protein synthesis. Its mechanism of action consists of binding to the 30S ribosomal subunit to block the aminoacyl-tRNA, an essential part of bacterial translation, thus preventing the spread of the bacterial infection. One family of enzymes, named Tetracycline destructases, are the primary group of enzymes that target Tetracycline to render the antibiotic ineffective. These flavoenzymes function as monooxygenases and use reduced flavin adenine dinucleotide (FADH2) and molecular oxygen to covalently modify the Tetracycline scaffold, rendering it inactive. Unlike other mechanisms of antibiotic resistance, the Tetracycline destructase family completely destroys the antibiotic molecule to an irreversible end. The tetX gene codes for the original NADPH-dependent oxidoreductase, TetX, found to cause Tetracycline resistance in Staphylococcus aureus, Escherichia coli, and other gram-positive and gram-negative bacteria. A variant of this resistance enzyme, TetX(1), is a smaller, truncated version missing part of the N-terminal sequence, rendering it incapable of binding the flavin cofactor (FAD). This study aimed to engineer an evolved version of TetX(1), TetX(1)-EVD, which was designed to mimic TetX and contain the full N-terminal sequence for FAD binding. Analysis of TetX(1) versus TetX(1)-EVD protein structures using Alphafold2.0 technology confirmed the missing N-terminus in TetX(1) and added FAD domain in the TetX(1)-EVD construct. Molecular cloning techniques including polymerase chain reaction (PCR), restriction digests, and ligations were employed to insert the new gene construct into the pBAD24 expression vector. Both the tetX(1)-pBAD24 and tetX(1)-EVD-pBAD24 plasmids were transformed into chemically competent E. coli cells to generate working laboratory strains. These strains were used to compare the effectiveness of TetX(1) and TetX(1)-EVD in conferring resistance against Tetracycline antibiotics. Cell-based activity experiments, including minimum inhibitory concentration (MIC) tests, confirmed expected Tetracycline and Doxycycline resistance conferred by TetX, and less noticeable amounts of resistance from TetX(1) and TetX(1)-EVD. Collectively, these findings confirm the lack of the complete N-terminal sequence in TetX(1) to be a significant hindrance in generating antibiotic resistance, suggesting the possibility that the catalytically active TetX could have evolved from the inactive truncated TetX(1).

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