Fig. 5

CRISPR/Cas-based protocol for simultaneous two-gene deletions E. coli BL21(DE3)∆ompA(pCasRed) was co-transformed with either 100 ng pCRISPR-ompF_5′G-ompF_3′I plasmid and the two dDNAs ompF_5′G-120-∆30 and ompF_3′I-120-∆30 (10 μg each) (a) or with 100 ng pCRISPR-lpp_B-fecA_B and lpp_B-120-∆30 and fecA_B-120-∆30 dDNAs (10 μg each). b Transformant colonies were selected on LB agar plates supplemented with 25 μg/ml Cm and 50 μg/ml Km. Colony PCR was carried out using two different couple of primers to screen each genomic locus (indicated at the bottom of each gel) on a randomly selected number of colonies and the PCR products separated on 2% agarose gels. Asterisks indicate those colonies in which deletion occurred at both gene loci. The primer sequences used for PCR experiments are reported in Additional file 1: Table S5