Fig. 6

pCRISPR-SacB-gDNA plasmid curing using 5% sucrose containing medium. A single colony from E. coli BL21(DE3)ΔompA strain carrying both the pCasRed (Cm resistance) and pCRISPR-SacB-gDNA (Km resistance) was grown at 37 °C in LB-medium containing 5% sucrose and 25 μg/ml Cm. After 14 h growth, 100 μl of culture were plated on LB-agar plates containing either Cm (25 μg/ml) + Km (50 μg/ml) or Cm (25 μg/ml) alone. The loss of pCRISPR-SacB-gDNA plasmid was verified by 1.5% agarose gel analysis of plasmids extracted from bacteria directly collected from the Cm-containing agar plate. As control, the same colony was grown in the absence of 5% sucrose and plasmid extraction was carried out from bacteria collected from LB-agar plate containing 25 μg/ml Cm