Fig. 4

Representation of the stepwise approach used to isolate strains carrying multiple mutations. Day 1: E. coli BL21(DE3)∆ompA(pCasRed) was co-transformed with 1 μg/ml of pCRISPR-SacB-fecA_B and 10 μg/ml of donor fecA-120-∆30nt and transformant colonies were selected on LB agar plates supplemented with Cm (25 μg/ml) and Km (50 μg/ml). Day 2: Ten colonies were randomly selected and screened by PCR using primers designed to generate DNA fragments from mutated colonies of 200 bp. PCR products were analyzed on 2% agarose gels. One mutant clone was subsequently inoculated into 5 ml of LB supplemented with 5% sucrose and 25 μg/ml Cm. Day 3: The overnight culture was used to prepare competent cells, which were subsequently co-transformed with 1 μg/ml of pCRISPR-SacB-lpp_B and 10 μg of double strand donor DNA lpp-120-Δ237. Day 4: Ten colonies were randomly selected and screened by PCR using primers designed to generate DNA fragments from mutated colonies of 400 bp. PCR products were analyzed on 2% agarose gels