Fig. 3

Overall scheme for L4A deletion using two systems in L. lactis 9k. (1) Plasmid pNZ5319ΔL4A or pNZ5417ΔL4A was first loaded into L. lactis 9k-3 cells and the recombinants were selected on M17 plates supplemented with 5 μg/mL chloramphenicol (Cm); for pNZ5417ΔL4A, 40 μg/mL X-gal and 10 IU/mL nisin were also added; (2) recombinants harboring plasmid pNZ5319ΔL4A or pNZ5417ΔL4A were cultured in M17 medium supplemented with 5 μg/mL chloramphenicol for generations, and positive mutants with successful L4A deletion were selected by replica plating method (for “pNZ5319/pNZTS-Cre gene deletion system”) or color change (for “new gene deletion system”); (3) deletion of the chloromycetin resistance marker and elimination of temperature-sensitive plasmid pNZTS-Cre [19]