Bacteriophage are viruses that infect prokaryotes. Although they have a vast population, their genetics and regulation are largely unknown. For example, most phage currently sequenced have functions predicted for approximately one third of their genes. A fascinating place to begin with is knowing which genes specifically kill their bacterial hosts, or are cytotoxic. Knowing the cytotoxicity of each phage gene individually would give us a better understanding of phage genomes, and phage-host interactions. Four genes (gp1, gp3, gp4, and gp37) from a phage named Cepens, will be amplified using Polymerase Chain Reaction (PCR) and cloned into a plasmid (pExTra) with isothermal assembly. This cloned plasmid will then be chemically transformed into E. coli and transformants verified through Colony PCR and selective Kanamycin plates. These bacteria can mass produce the plasmid and allow it to be extracted using a mini-kit for future molecular usage. Using electroporation, the cloned plasmid with the gene of interest will be introduced into a known bacterial host for Cepens, Mycobacterium smegmatis mc2155. Cytotoxicity assays will be performed using the inducer anhydro-tetracycline (aTc) and measured with bacterial growth where decreased growth indicates cytotoxicity of that individual gene. Genes 1, 3, and 4 have annotated functions as a helix-turn-helix DNA binding protein, Helix-turn-helix domain protein, and unknown respectively. We would expect genes 1 and 3 to not be cytotoxic as these functional predictions indicate roles in gene regulation, while 4 is unknown. Gene 37 has an annotated function of a putative Holin protein. We expect to see this as a cytotoxic gene given it is well characterized in the literature to be responsible for interfering with membrane stability and causing cell lysis. Knowledge of cytotoxicity of individual phage genes could have implications in therapeutics for development as potential drugs that can kill bacteria independently of phage infection limitations.

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