Figure 1.
The scheme of suicide gene-based counter-selection system plasmid and temperate phage recombination procedure. (A) Target and PAM sequence of blaNDM-1 was integrated with the suicide gene SacB in plasmid pSTK. (B) Integration of the CRISPR/Cas9 system within the temperate phage genome. MG1655 cells were lysogenised with the vB_365 prophage, and pKD46 and pSTk were subsequently transformed into this strain. The linear dsDNA recombination donor was prepared and delivered by electro-transformation. Transformants were screened on sucrose plates. Cells that recombined with CRISPR/Cas9 could survive in the presence of the activated suicide gene. vB_Cas9 was induced and harvested from the bacterial cells.