Beta-Galactosidase-Based Promoter Activity Assay

The activity of the cloned promoters was first evaluated on the plate. Briefly, day cultures of S. aureus USA300 strains harboring the plasmid constructed above were prepared by inoculating 100 μL of the overnight culture into 10 mL of TSB medium. After the OD₆₀₀ value reached 0.6, 5 μL of the bacterial culture was pipetted out and dropped onto a TSA agar plate containing 25 μg/mL chloramphenicol and 200 μg/mL X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). The plate was incubated at 30°C, and the color of the colony was monitored and photographed after 48 h.

To further quantify the promoter activity, analysis of beta-galactosidase activity using O-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate was performed and modified as previously described (Zhu et al., 2020). A single blue colony on the TSA X-Gal plate was inoculated into chloramphenicol-containing TSB medium overnight culture preparation. The next day, the day culture was prepared as described above. After 2, 6, or 10 h of cultivation, 100 μL of the bacterial culture was collected for OD₆₀₀ value measurement in a 96-well plate by using a Synergy H1 microplate reader (Vermont, United States).

Meanwhile, bacterial cells were harvested from the 200 μL culture at each time point by centrifugation at 5,000 g for 10 min at 4°C. The cells were resuspended in 100 μL of lysis buffer (60 mM K₂HPO₄, 40 mM KH₂PO₄, 100 mM NaCl, 0.1% Triton X-100, 50 μg/mL lysostaphin) for total protein extraction. The suspension was incubated at 37°C until the bacterial cells had completely lysed. Then, 50 μL of the lysate was pipetted onto a 96-well plate for OD₄₂₀ measurement. After that, 100 μL of a reaction buffer (60 mM K₂HPO₄, 40 mM KH₂PO₄, 100 mM NaCl, 0.1% Triton X-100, 5 mg/mL ONPG) was added to each well and mixed with the cell lysate. The 96-well plate was incubated in the Bioreader at 37°C with constant shaking, and the OD₄₂₀ was measured every 5 min. The slope of the linear part of the spectrophotometric output was used to calculate the specific activity as follows: Miller units = (1,000 × slope)/(V × OD₆₀₀), where V is the volume (in milliliters) of the culture used in the assay. This assay was repeated three times for each strain.