Investigating the Cytotoxicity of Bacteriophage Cepens Genes in Host Mycobacterium Smegmatis

Bacteriophage are viruses that infect prokaryotes. Despite their vast population, their genetics are largely unknown. For example, most sequenced phage have functions predicted for approximately one third of their genes. Fascinating data could be knowing which genes kill their bacterial hosts. Knowing the cytotoxicity of each phage gene would give 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 PCR and cloned into a plasmid (pExTra). This cloned plasmid will then be chemically transformed into E. coli and transformants verified through Colony PCR. From here the plasmid will be extracted for future molecular usage. Using electroporation, the cloned will be introduced into Mycobacterium smegmatis mc²155. Cytotoxicity assays will be performed using the inducer anhydro-tetracycline and measured with bacterial growth where decreased growth indicates cytotoxicity of that 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 literature to be responsible for interfering with membrane stability and causing cell lysis. Knowledge of cytotoxicity of individual genes could have implications in therapeutics for development as potential drugs that kill bacteria independently of phage infection limitations.