Fig. 2: Engineering of virulent E. coli, Enterococcus and Klebsiella reporter phages.
From: Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine
a Schematic representation of the nanoluciferase gene (nluc) insertion sites in the genomes of all reporter phages. For E. coli and Enterococcus phages, codon optimized nluc (516 bp + RBS) was incorporated immediately downstream of the major capsid gene (cps), whereas for Klebsiella phages, nluc was inserted downstream of the prohead assembly protein (gp167 for K1 and gp168 for K4). b–d Engineering method for virulent Enterococcus phages combining homologous recombination with CRISPR-Cas9-assisted counterselection. b Left: Schematic representation of the pSelect CRISPR locus used to restrict wildtype phage genomes by targeting two sequences flanking the nluc integration site. Right: Spot-on-the-lawn assays of 10-fold phage dilutions show plaquing efficiency on hosts with (+) or without (scr, -) CRISPR targeting. scr = non-targeting crRNA; - = no pSelect. c Schematic representation of the editing plasmids (pEditEfS3, pEditEfS7) used to incorporate mutated protospacer-adjacent motifs (PAMs) and nluc gene sequences downstream of the major capsid protein (cps) regions of EfS3 and EfS7 by homologous recombination and subsequent CRISPR escape of EfSs::nluc and EfS7::nluc candidates (counterselection) shown by spot-on-the-lawn assays of serial dilutions of phages after recombination. d 10 individual plaques were isolated from one replicate of data shown in Fig. S2b, c. Plaques were validated genetically and functionally using PCRs with primers P19/20 and P21/22 and RLU determination from individual plaques, respectively. HAL = homology arm left; HAR = homology arm right; put. cds = putative coding sequence; mtp = major tail protein; RLU = relative light unit. Source data are provided as a Source Data file.
