pSIM6

In genetic engineering, traceless modification is a popular genome manipulation technique. The rigor of the counter-selection system determines the efficiency of traceless modification. Studies have shown that a new type of counter-selection system kil, when combined with plasmid pSim6, has higher counter-selection stringency than pKD46 plasmid, indicating that the combination of kil and pSim6 plasmid can select recombinants more efficiently. Accordingly, four different non-essential gene loci (lacI, dbpa, ack and glk) in E. coli strains W3110, MG1655 and DH10B were analyzed. The tet/kil pre-inserted into these sites are genetically replaced with exogenous gene fragments of different lengths.

The results showed that the recombination efficiency of kil was significantly improved when combined with pSim6 plasmid compared with its combination with pKD46 plasmid, and the effect was more obvious with the growth of exogenous fragments. When the exogenous fragment is 1 000 bp, kil combined with pSim6 plasmid is 1.2-2 times more efficient than its combination with pKD46. When the exogenous fragment is 2 000 bp, it is 2.2-5 times that of pKD46. In summary, the combination of kil and pSim6 has higher recombination efficiency and is more conducive to traceless modification of the E. coli genome, which provides more options for E. coli recombination engineering.

Figure 1. Analysis of recombination efficiency at glk locus. (A) Identification of recombinants harboring tet/kil by PCR. (B) PCR detection of tet/kil substitution using plasmid pSim6. (C) Positive rate of tet/kil substitution using plasmid pSim6. (Li, Yujuan, et al., 2019)