A synthetic biology approach to assemble and reboot clinically-relevant Pseudomonas aeruginosa tailed phages

In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically-relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination (TAR) cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step towards the construction of clinically-relevant, engineered P. aeruginosa phages.