Engineering φX174 to create a biocontained phage therapy model

Antibiotic resistant bacteria are an increasing concern amongst hospitals worldwide, with many patients presenting infections that are nonresponsive to current treatments. To resolve this problem, many scientists are turning to phage therapy for treatment. Unfortunately, phage therapy harbours drawbacks including fear of gene transfer between different microbes within human microbiomes, and the potential for off-target interactions interrupting other microbial niches within the body. Engineered phage may reduce some of these drawbacks, creating phage with broader host range or the ability to degrade biofilms, but to realise these benefits outside the lab, engineered phage need to show they cannot spread widely in the environment. In this thesis I created a biocontained phage therapy model to attempt to address these concerns. In employing the non-canonical amber start codon (TAG) at the beginning of φX174 capsid genes, I engineered a phage that could only replicate when provided with the mutant initiator tRNA_CUA. As a result, I was able to provide a method of biocontainment which maintained phage stability, maintained its ability to adsorb to host cells, and prevented it from replicating, although host-killing efficiency was reduced from wild-type phage. This study provides a platform technology for the effective containment of bacteriophages and takes the first step towards enabling phage therapy to become an accessible treatment option.