Generation of E. coli Deletion Strains

Plasmid and genomic locus maps are provided in Supporting Information file 1.

Individual genes were deleted from the E. coli genome using CRISPR-Cas9-assisted recombineering as described by Jiang and co-workers.⁴⁵ For a given locus, we chose the best validated guide RNA corresponding to Guo et al.,⁴⁶ and cloned it into a modified pTargetF plasmid using Gibson assembly (New England Biolabs, USA). The plasmid, pTargetF_sacB, carried the Bacillus subtilissacB gene which confers susceptibility to sucrose and enables counterselection against the plasmid. The exact deletion was encoded by the ssDNA oligo.⁴⁷ The editing protocol was as follows: an E. coli carrying pCas plasmid was inoculated in a 2 mL SOB medium supplemented with kanamycin in a culture tube and grown overnight at 30 °C, 220 rpm. This culture was diluted 50-fold into a 3 mL fresh SOB medium (0.5% (w/V) yeast extract, 2% (w/V) tryptone, 10 mM sodium chloride, 2.5 mM potassium chloride, and 20 mM magnesium sulfate. SOC medium is SOB medium supplemented with 20 mM glucose. If indicated, antibiotics were added in the following concentrations (mg L^–1): 50 kanamycin, 20 streptomycin sulfate) supplemented with kanamycin and grown to mid-exponential phase (OD₆₀₀ 0.4–0.7). At this point, expression of recombineering enzymes was induced with 10 mM l-arabinose, and the culture was incubated for 27 min. 2 mL of the culture was harvested by centrifugation (4 °C, 5000g, 2 min) and made electrocompetent by washing five times with 1 mL of ice cold 10% (V/V) glycerol. 100 ng of pTargetF_sacB plasmid and ssDNA oligo to a final concentration of 5 μM were added to the electrocompetent cells and electroporated using a Bio-Rad MicroPulser Electroporator (Bio-Rad, Switzerland). Subsequently, cells were recovered in a 1 mL SOC medium for 2 h and streaked out on an agar plate containing LB medium supplemented with kanamycin and streptomycin which was incubated overnight at 30 °C. Successful deletion of a locus was confirmed by colony PCR. pTargetF_sacB plasmid was cured by streaking out strains on agar plates containing LB medium supplemented with 10% (w/V) sucrose and kanamycin and incubated overnight at 30 °C.