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THE CYTOTOXIC EFFECT OF INSERTED BACTERIOPHAGE GENES ON MYCOBACTERIUM SMEGMATIS

Abstract

The overprescription of antibiotics has contributed to the rise of multidrug resistant bacteria. Bacteriophages, viruses that can specifically target these bacteria, have been historically proven useful in fighting bacterial infections. While penicillin reduced Western interest in phage therapy, limited antibiotic access led the Soviet Union to continue its development. Currently, the west is returning to the research. The study of bacteriophage and their genetics 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 to treatment of multiple drug-resistant infections. In this study, the genome of bacteriophage Cepens, discovered at the University of North Georgia, was analyzed. Various biotechnological techniques, including PCR, gel electrophoresis, protein purification, and transformations, were employed to investigate the viral genome for cytotoxic genes. Host bacteria containing the absorbed pExTra vector plasmid were selected for by the use of kanamycin supplemented media. Mycobacterium smegmatis served as a model host due to its genetic similarities with Mycobacterium tuberculosis, a pathogen which has developed drug resistance. A plasmid containing the viral gene of interest was inserted into Escherichia coli through a heat shock protocol, then further introduced into M.smegmatis via electroporation. The model host amplifies and expresses the plasmid alongside their own genetic material. Gene induction, subsequent colorimetric assays and growth kinetics were used as data to inform which Cepens genes were toxic to M. smegmatis. Our results indicate potential cytotoxic effects on M. smegmatis through bacteriophage Cepens’ genes 11 and 13 based on cytotoxic assays with control variables. Visual confirmation of abnormal colonies on the bacterial serial dilution series suggests cytotoxic results.

Acknowledgements

University of North Georgia Department of Biology Department, University of Georgia Electron Microscopy

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