1. Synthetic perturbation of DE and DV genes.
Synthetic perturbation of DE and DV genes. (A) CRISPR interference (CRISPRi) is used to repress gene expression by blocking progression of RNA polymerase (RNAP) at a site specified by the sequence of the sgRNA. The dCas9 protein and the sgRNA are expressed from a medium-copy-number plasmid. (B) MIC was determined for individual colonies from each CRISPRi strain. Colonies were grown for 16 h in a range of antibiotic concentrations, and MIC was determined visually through a resazurin assay. (C and D) The MICs of ampicillin (C) and gentamicin (D) are shown for individual colonies from each CRISPRi strain. Box plots show the interquartile range. The median is marked with a horizontal line. Whiskers demarcate minimum and maximum values. Individual data points are overlaid on the box plots. n = 19 to 50 colonies per strain. (E) Representative plates from swarming motility assay, for E. coli BW25113 wild-type and five knockout strains after 48 h of growth. (F) Average area from swarming motility assay. Error bars represent the standard deviation across n = 3 replicates per strain. (G) Relative mutation rates for CRISPRi strains (rate of strain/rate of RFP-i control). Error bars represent the standard deviation (n = 30 parallel cultures for each). (H) Resazurin reduction curves. RFU, relative florescence units. Error bars are the standard deviation (n = 4 replicates). gent, gentamicin. (I) Slopes of resazurin reduction curves in panel H. For panels F, G, and I, asterisks indicate a result significantly different from the control (P < 0.05).
Keesha E. Erickson et al. mSphere 2017; doi: /mSphere