GFP assay

The pLNRK-GFP plasmid was generated by removing the intron from pLNRK-RIG via PstI and SpeI digestion, followed by the ligation of the green fluorescent protein (GFP) gene that was amplified from pHGSap [69] using NEBuilder (IDT6054 and IDT6055). Individual mutants were cured, as described above, and transformed with pLNRK-GFP by electrotransformation, followed by PCR confirmation (IDT6056 and IDT6057). Plasmid-bearing strains were grown and subcultured 1:10 in 40 mL of GM17 Cam₁₀. After 2 h, the cultures were split into two tubes. Half of the cultures were induced for GFP with 10 ng/mL nisin for 4 h, the others remained uninduced, and growth continued. One mL taken every hour was transferred to a 96-deepwell plate (Eppendorf), centrifuged at 3220 x g for 8 min, washed, and serial diluted in 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). Volumes of 200 μL were transferred to a 96-well plate (clear flat bottom, Corning) to measure OD₆₀₀ and GFP fluorescence (black flat bottom, Corning) on a plate reader (BioTek Synergy H1). Data were analyzed using BioTek Gen5 and Microsoft Excel.