Abstract

With the increasing antibiotic resistance of pathogenic bacteria, we are entering the post-antibiotic era with no antibiotics available. Phage therapy is one of the promising strategies to fight against pathogenic bacteria. Synthetic biology provides the feasibility to engineer natural phages with desired properties suitable for phage therapy. Here, we are developing a yeast-based platform to genetically manipulate and reboot Coliphage T4, whose 168.9 kbp-sized genome is too huge to manipulate through existing methodologies. The whole T4 genome is amplified and assembled hierarchically through overlapping extension PCR, yeast transformation associated assembly, and CRISPR/Cas9-facilitated homologous recombination assembly. The reconstituted T4 genome is then electroporated into E. coli to generate phage particles. We expect that our platform is suitable for the manipulation of huge phages with large-sized genomes.