Chemidoc mp imaging machine

Lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100 and protease inhibitors) was used to lyse the confluent cells after three ice-cold PBS washes. The lysates were centrifuged for 15 min at 13,000× g, and the protein concentrations of the supernatants were measured by a Pierce™ BCA Protein Assay Kit (Thermo Scientific™, Dreieich, Germany). We used 12% TGX gels Stain Free gels (Bio-rad, Feldkirchen, Germany; Cat. 1610181) to separate the proteins before blotting them to nitrocellulose membranes 0.2 µM through a Trans-Blot Turbo Transfer System (Bio-rad, Feldkirchen, Germany). Identifying the proteins was carried out by fluorescence antibodies StarBright Blue 520 and 700 (Bio-rad, Feldkirchen, Germany) using the ChemiDoc MP imaging machine (Bio-Rad, Feldkirchen, Germany). ImageJ software provided by the National Institutes of Health, Bethesda, MD, USA, was used to assess the gray density of Western blots. The signal for the protein of interest is normalized to the total amount of protein loaded into the lane using a stain-free membrane [47 (link)].

Gradient plates were prepared as previously described (Bryson and Szybalski, 1952 ) with slight modifications. In brief, a 100 mm x 100 mm x 1.5 mm square petri dish (Thomas Scientific) was propped up on a 1 cm ledge to create a slant. 40 ml of LB with 1.5% agar and either 10 μg/ml chloramphenicol or 300 μg/ml erythromycin was poured and allowed to harden. The plate was then laid flat and 40 ml of LB with 1.5% agar was overlaid. For strains carrying plasmids, both top and bottom agar were also supplemented with 50 μg/ml carbenicillin and 0.02% arabinose. Colonies for each strain isolated from sterile LB-Agar plates were grown overnight in LB (with carbenicillin for plasmid-containing strains) at 37°C with shaking at 250 rpm. The overnight cultures were diluted 1:100 and grown in the same media at 37°C with shaking at 250 rpm to exponential phase (OD₆₀₀∼0.4). Aliquots (15 μl) were dripped down the plate starting from the edge with the lowest antibiotic concentration. The drips were allowed to dry, and the plates were incubated at 37°C for 16 h and imaged using the fluorescein setting on a ChemiDoc MP Imaging machine (Biorad). Each gradient plate assayed cultures generated from separate single colonies. Reproducible results were observed for 10 gradient plate assays conducted over three separate days. A representative image was selected for publication.