Ni protein assay

Bocillin labeling of PBPs was performed as described previously (Cho et al., 2016 (link)) with the following modifications. After incubation with Bocillin washing the cell pellets 3X with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄), the pellets were frozen at −80°C. Cell pellets were then thawed, resuspended in 1 mL of PBS and lysed using a FastPrep-24 (MPBio). Cells were lysed using matrix B in 2 mL tubes for 40 s at 6 m/s. Cells were centrifuged for 3 min at 7,000 rpm to remove the undisrupted cells and matrix and the supernatant was transferred to a fresh tube. Membranes were then pelleted by ultracentrifugation at 100,000 × g for 20 min at 4°C. The membrane pellets were then washed with 1X PBS and resuspended in 60 μL 1X PBS. Resuspended samples were mixed with 60 μL 2X Laemmli sample buffer (100 mM Tris-Cl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 5% (v/v) β-mercaptoethanol) and boiled for 10 min at 95°C. After measuring the total protein concentration of each sample with the NI-protein assay (G-Biosciences), an equivalent amount of total protein for each sample was then separated on a 10% SDS-PAGE gels. Bocillin-labeled proteins were then imaged using a Typhoon 9500 fluorescence imager (GE Healthcare) with excitation at 488 nm and emission at 530 nm.

Cultures of MG1655 and SP10 (Δtol-pal) strains were grown to an exponential phase (OD₆₀₀ between 0.4 and 0.5) in LB at 30°C. Aztreonam or mecillinam was added to 15 mL cultures at the final concentrations of 0, 0.1, 1, or 10 μg/ml and incubated for 5 min at 30°C. Cells were then collected by centrifugation at 4°C, washed with ice-cold 1X PBS twice, resuspended in 500 μl 1X PBS supplemented with 10 mM EDTA and 15 μM Bocillin (Invitrogen), and incubated at room temperature for 15 min. After incubation, the cell suspensions were washed with 1X PBS once, resuspended in 500 μl 1X PBS, and disrupted by sonication. After a brief spin for 1 min at 4 Krcf to remove undisrupted cells, membrane fractions were pelleted by ultracentrifugation at 200 Krcf for 20 min at 4°C. The membrane fractions were then washed with 1X PBS and resuspended in 50 μl 1X PBS. Resuspended samples were mixed with 50 μl 2X Laemmli sample buffer and boiled for 10 min at 95°C. After measuring the total protein concentrations of each sample with the NI-protein assay (G-Biosciences), 25 μg of total protein for each sample was then separated on a 4–20% gradient SDS–PAGE gel and the labeled proteins were visualized using a Typhoon 9400 fluorescence imager (GE Healthcare) with excitation at 488 nm and emission at 520 ñm.