Optimization of CRISPR-guided platforms for phage genome engineering in Pseudomonas aeruginosa
Bacteriophage (phage) infection has significant impact on host bacterial physiology, which involves the phage-encoded elements. Despite the importance and versatility of the phage-encoded elements in both research and applications, most of the phage elements are poorly characterized mainly due to the lack of systematic platforms for genome engineering. We previously generated a designer phage engineering platform in the opportunistic human pathogen, Pseudomonas aeruginosa, by coupling the Streptococcus pyogenes Cas9 (SpCas9) and the Red recombination system of the coliphage λ (λRed). In this study, we optimized the platform by replacing an isopropyl β-D-1-thiogalactopyranoside-inducible promoter with a crystal violet-inducible promoter, pJExD, under sufficiently tight regulation by its codon-optimized repressor, EilR. The gene(s) for the single guide RNA and the editing template have been cloned in a multicopy plasmid. Topics discussed will include the updated results regarding creation of the designer phage particles with the major coat protein tagged with either a fluorescent protein or antimicrobial peptides.
2025 Spring Convention