An efficient, scarless, selection-free technology for phage engineering

Abstract

Many phage engineering technologies, mainly based on the CRISPR-Cas system, have been recently developed. Here we present a method to genetically engineer the Escherichia coli phages T5, T7, and P1 by adapting a technology, called pORTMAGE, developed for engineering bacterial genomes. The technology comprises of E. coli harboring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, ranging from 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We demonstrated the use of this technology for single-base substitutions, for deletions of 50 bases, for insertions of 20 bases, and for three different phages. The technology may be adjusted for use across many bacterial and phage strains.

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“…Constructing T7 phage mutants: T7 mutants were constructed using the pORTPHAGE method 69 , a MAGE 70,71 -based system for the mutagenesis of bacteriophages. Briefly, E. coli K-12 strain harboring the pORTMAGE-Ec1 plasmid was grown to reach early log phase (OD600 ~0.3).…”