4 methyl umbelliferyl β d galactopyranoside mug

Cells carrying the plasmid pCL-P1-lacZ [7 (link)] were grown and collected at a desired time point by centrifugation. The collected cells were washed with AB buffer (60 mM K₂HPO₄, 40 mM KHPO₄, 100 mM NaCl, pH 7.0), suspended in 100 μl of AB buffer, and mixed with 5 μl of lysostaphin (2 mg/ml). After 15 min incubation at 37°C, the samples were mixed with 900 μl of AB buffer containing 0.1% (v/v) Triton X-100, and the β-galactosidase assay was performed at room temperature. As a substrate, 4-methyl umbelliferyl β-D galactopyranoside (MUG, Sigma) was used in the hydrolysis reaction, which was read at 366 nm excitation and 445 nm emission wavelengths.

CWBIs and negative control compounds were either from Sigma–Aldrich or the sources listed in Table S2. Susceptibility testing²⁹ was used to define appropriate concentrations of control compounds for assay. Biosensor strains were cultured in tryptone soya broth (TSB; Oxoid) at 37°C with vigorous aeration to an OD₆₀₀ of 0.2 and challenged with antimicrobial agents for 60 min. In the case of biosensor constructs in Ts mutants, strains were grown at 30°C to an OD₆₀₀ of 0.2, before shifting the temperature to 42°C for 60 min. Post-challenge, OD₆₀₀ was measured to allow changes in cell density to be accounted for in calculating biosensor induction. An aliquot of culture (typically 200 µL) was centrifuged, and the washed cells resuspended in 0.5 volumes of AB buffer^{30 (link)} containing lysostaphin (15 mg/L) and the fluorogenic β-galactosidase (β-gal) substrate, 4-methylumbelliferyl β-D-galactopyranoside (MUG, 500 mg/L; Sigma–Aldrich), and incubated at 25°C with shaking for 90 min. Production of β-gal was determined as described.^{30 (link)}