Fig. 5

A general procedure for Lp_0640-41-42-mediated genome engineering. (1) A mutagenesis vector containing a lox66-cat-lox71 cassette flanked by 1-kb homologies was constructed. Homology arm A and Homology arm B were generated by PCR of the upstream and downstream of the target locus in the genome. DNA fragments sharing terminal sequence overlaps (indicated as colored-squares) were assembled via Gibson method. (2) dsDNA substrate was generated from the mutagenesis vector by PCR and subjected to DpnI digestion to eliminate the plasmid template. (3) Lp_0640-41-42-mediated recombination was performed to edit the genome of strains and correct mutants could be screened out by antibiotic selection and PCR test. (4) The recombinase-expressing plasmid was eliminated by culturing a correct mutant derived in procedure 3 in the absence of erythromycin selection for 24 h. (5) The Cre helper plasmid was used to excise the cat marker, and a lox72 site (34 bp) that is poorly recognized by Cre was formed. (6) The Cre plasmid was eliminated by culturing a correct mutant derived in procedure 5 in the absence of erythromycin selection for 24 h. The resultant strain was free of any plasmid or genetic selection marker