How to edit the genes of nature's master manipulators BerkeleyLab NatureMicrobiol
) was grown overnight in LB+Ch media at 37 °C and 250 r.p.m. Strains were diluted into fresh media to an ODc.f.u. per well. ‘HR phage lysate’ was added to each well to achieve an MOI of 0.01 . Infection was monitored in a Biotek Cytation 5 plate reader at 200 r.p.m. shaking at 37 °C, with ODreadings every 5 min. Infection was allowed to proceed until there was a visible population crash .
To confirm the genotype of edits, we performed unbiased PCRs followed by Sanger sequencing. In addition, for gene deletions we performed N-terminal and C-terminal PCRs. Primers for unbiased PCRs were designed to amplify from the phage genome 150–300 bp outside of the UP and DN homology arms supplied from the editing vectors. For N-terminal and C-terminal PCRs, reverse and forward facing primers touchdown on the small primer binding site provided on HR vectors .
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