3.6. Construction of Nirsir Complementation and Overexpression Mutant Strain of JD-014

For the construction of nirsir complementation and overexpression of mutant strains, a fragment of nirsir was amplified from JD-014 genomic DNA and then cloned into the vector pMA5 with the ClonExpress^® II one-step cloning kit (Vazyme, Nanjing, China). The recombinant plasmid pMA5-nirsir was confirmed by sequencing and transformed into competent cells of JM109 for overnight cultivation in LB ampicillin solid medium at 37 °C. Positive clones were subsequently transformed into JD-014 and the mutant JD-014Δnirsir strain respectively through electro transformation, and selected in LB plates with 30 μg/mL kanamycin at 37 °C for 12 h. The complementation and overexpression mutants were verified by PCR with primers of pMA5-a-F/pMA5-a-R.