Abstract:
Anti-Microbial resistance amongst many strains of pathogenic organisms is a major issue for the healthcare sector. Phage therapy has come about as a novel method against certain antibiotic resistant strains. However, lysis due to phages can release intact DNA into the surrounding which can pass off antibiotic resistance to new organisms through Horizontal Gene Transfer. SauUSI is a Type-IV Restriction Endonuclease that has the ability to specifically target methylated DNA. Unlike other DNA-cutting enzymes, SauUSI cuts at multiple sites between its recognition sites which shreds DNA into multiple, short fragments. In this study, we have aimed to engineer a T7 bacteriophage that contains the gene which codes for SauUSI. The T7 phage acts as a delivery system that can provide SauUSI directly inside the cell and destroy any methylated DNA. We have shown that SauUSI can easily shred methylated genomes to small DNA fragments and the presence of SauUSI inside the cell slows down its growth. Using the technique of in vitro fragment generation, assembly and rebooting using electroporation, we have been able to generate edited T7 phages. Such a system can be used as an alternative for antibiotics without the risk of HGT, as the combined action of SauUSI and lytic phages will lyse the cell and destroy its DNA.
