Quantitative β-Galactosidase Activity Assay

The β-galactosidase assays were modified from a previously reported study [65 (link)]. Overnight bacterial cultures were diluted 1:500 in TSB. If needed, appropriate amounts of oxidants, iron chelators, or FeCl₃ were included in the medium. The β-galactosidase activity was monitored by collecting samples at different time points. A total of 100 µL of bacterial culture was added to 900 µL of Z Buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0, 0.2% β-mercaptoethanol). A total of 1 µL of 0.1% sodium dodecylsulfate (SDS) and 50 µL of chloroform were added to the suspension, which was mixed vigorously for 20 s. The suspension was then incubated for 5 min at 30 °C. A total of 200 µL of 4 mg/mL 2-nitrophenyl β-d-galactopyranoside (ONPG, Sigma) was added to the cells. The reaction was stopped by adding 500 µL of 1 M Na₂CO₃. The suspension was centrifuged at 14,000× g for 3 min, and the optical densities of the supernatant were read at 420 and 550 nm. The β-galactosidase activity was then calculated in Miller Units (MUs) according to the following equation: $$\mathrm{MU}=\frac{1000\times \left({\mathrm{OD}}_{420}-1.75\times {\mathrm{OD}}_{550}\right)}{\mathrm{Time}\left(\min \right)\times \mathrm{Volume}\left(\mathrm{mL}\right)\times {\mathrm{OD}}_{600}}$$