PHEIGES: all-cell-free phage synthesis and selection from engineered genomes

Antoine Levrier, Ioannis Karpathakis, Bruce Nash, Steven D. Bowden, Ariel B. Lindner () and Vincent Noireaux ()
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Antoine Levrier: University of Minnesota
Ioannis Karpathakis: University of Minnesota
Bruce Nash: Cold Spring Harbor Laboratory
Steven D. Bowden: University of Minnesota
Ariel B. Lindner: Center for Research and Interdisciplinarity
Vincent Noireaux: University of Minnesota

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: Abstract Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

Date: 2024
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DOI: 10.1038/s41467-024-46585-1

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