Characterization of Bacteriophage genes impacting Mycobacterium smegmatis via plasmid (pExTra) transformations.

The overprescription of antibiotics has contributed to the rise of multidrug-resistant bacteria. Bacteriophages, viruses that specifically target these bacteria, have been historically proven useful in fighting bacterial infections. However, following the discovery of Penicillium notatum and its antibacterial properties, Western interest in phage therapy declined, while countries limited to antibiotic access, such as the Soviet Union, continued to explore its therapeutic potential. In this study, the genome of bacteriophage Cepens, discovered at the University of North Georgia by the Freshman Discovery Lab, was utilized. Various biotechnological techniques, including PCR, gel electrophoresis, protein purification, and transformations were employed to investigate the viral genome for cytotoxic genes. Cytotoxic genes target bacterial hosts by disrupting essential cellular functions.

To access phage-driven cytotoxicity, Mycobacterium smegmatis, serves as a model host due its genetic mycobacterial similarities with Mycobacterium tuberculosis, a well-known pathogen. A plasmid containing the viral gene of interest (pExTra) was introduced into M.smegmatis via electroporation, while Escherichia coli served as a secondary model organism, with the plasmid instead being introduced through heat shock. Supplemented media plates hold the light-sensitive antibiotic kanamycin, that provides the selection of bacterial colonies carrying pExTra. The model hosts culture the plasmid alongside their own genetic material.

Our results indicate genes that are capable of effectively compromising M.smegmatis based on dilution colony assays. This has the potential to be particularly useful in a clinical setting in cases where pathogenic bacteria are no longer affected by antibiotics. Once identified, corresponding protein sequences can be placed into databases to provide future physicians with alternative options.