Plaque assays

To assess the activity of various AcrF1 mutants in vivo, the pHERD30T plasmid was used to transform P. aeruginosa strain PA14. The PA14 CRISPR–Cas system naturally targets phage DMS3m, preventing its replication, while phage DMS3 is insensitive to the CRISPR–Cas system. Phage lysates at a starting concentration of 10⁷ plaque-forming units per mL were spotted in 10-fold serial dilutions onto a lawn of PA14 containing empty vector, wild-type AcrF1, or mutant AcrF1 and plaquing efficiencies at 30 °C were determined for three separate experiments.