Attempts Towards Site-directed Mutation of an Endonuclease Gene and Insertion of a Gene in a Bacteriophage Genome

Abstract

Anti-microbial resistance is a growing issue for the healthcare sector, with more and more pathogenic strains propping up every day. It isn’t enough to just kill the cells, as the DNA released into the environment can still confer the resistance to neighbouring cells through Horizontal Gene Transfer (HGT), and hence there is the need to develop measures that can do a two-fold job: kill the resistant-strain cells and prevent further propagation. SauUSI is a Type-IV Restriction Endonuclease that has been shown to have a “shredding” activity in methylated dsDNA. It creates several cuts between recognition sites, essentially putting the DNA through a biochemical paper-shredder. In my thesis, I aimed to engineer a lytic phage delivery system for SauUSI. The phage is responsible for cell lysis while the SauUSI is meant to shred all of the cellular DNA, thus making it meaningless in the context of HGT. Using Gibson Assembly as a synthesis method, and subsequent phage rebooting, I tried to create these phage-SauUSI constructs. The second half of the thesis focussed on identifying potential residues responsible for the specific recognition sequence of SauUSI, and generating mutations in the protein to allow us to alter it. This would allow for a widening in the range of DNA that the construct can shred and have potential therapeutic applications.