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A fully synthetic Golden Gate assembly system for engineering a Pseudomonas aeruginosa phiKMV-like phage

Kortright, Kaitlyn1; Sikkema, Andrew2; Selvakumar, Hemaa3; Antani, Jyot1; Chan, Benjamin1; Davidson, Matthew1; Newman, Benjamin2; Potapov, Vladimir2; Silva-Valenzuela, Cecilia2; Hopkins, Max3; Tabatabaei, S. Kasra2; McBride, Robert3; Turner, Paul1; Lohman, Gregory2

Published Dec 04, 2025 on Dryad. https://doi.org/10.5061/dryad.pzgmsbd23

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Abstract

Bacteriophages have applications in biotechnology, including human and veterinary medicine, agriculture, food safety, and biosecurity. One example of resurging importance is phage therapy, the use of phages to treat antibiotic-resistant bacterial infections. Phage therapy currently requires screening of environmental phages against the infecting strains for each individual patient, a laborious process that limits the development of standardized treatments. To overcome limitations of narrow host range inherent to many native phages, a robust genomic engineering platform is required, which permits rapid and dependable genome production and engineering for non-model phage. Here we describe an engineering platform for a phiKMV-like Pseudomonas aeruginosa phage, 41S1, that builds on previous work in the rapid assembly of small genomes through one-pot, High Complexity Golden Gate Assembly (HC-GGA). This system divides the 41S1 genome into DNA fragments small enough to be conveniently synthesized and to avoid toxicity during DNA propagation, with all but one maintained in Escherichia coli. These fragments are readily assembled in a high-accuracy, one-pot reaction; phages can be rescued by direct transformation into P. aeruginosa PAO1 or E. coli 10-beta cells. We demonstrate the precise generation of point mutations, DNA insertions and deletions, and the addition of fluorescence reporter genes that are expressed during phage replication. All viable genotypes could be generated with a near 100% success rate with minimal screening. This system demonstrates the potential of HC-GGA for the rapid production and engineering of phages and provides a chassis for the development of phages that broadly target the opportunistic human pathogen P. aeruginosa.