Gel doc 2000 image analyzer

Recombinant Anabaena sp. PCC 7120 FurA protein was expressed in E. coli BL21 (DE3) (EMD Biosciences) and purified according to previously described methods [84 ]. The promoter regions of each gene of interest were obtained by PCR using the primers described in Table F in S1 File. Electrophoretic mobility shift assays (EMSA) were performed as described previously [34 (link)]. Briefly, 120–140 ng of each DNA fragment were mixed with recombinant FurA protein at concentrations of 300, 500 and 700 nM in a 20 μl reaction volume containing 10 mM bis-Tris (pH 7.5), 40 mM potassium chloride, 100 μg/ml bovine serum albumin, 1 mM DTT, 100 μM manganese chloride and 5% glycerol. In some experiments, EDTA was added to a final concentration of 200 μM. To ensure the specificity of EMSA, the promoter region of Anabaena sp. nifJ (alr1911) gene was included as non-specific competitor DNA in all assays [17 (link)] The amount of competitor DNA (90–140 ng) was optimized in previous assays in order to detect unspecific binding of FurA without great molar excess. Mixtures were incubated at room temperature for 20 min, and subsequently separated on 4% non-denaturing polyacrylamide gels in running buffer (25 mM Tris, 190 mM glycine) at 90 V. Gels were stained with SYBR^® Safe DNA gel stain (Invitrogen) and processed with a Gel Doc 2000 Image Analyzer (Bio-Rad).

The DNA binding activity of the recombinant HsrA response regulator as well as its putative inhibition by HsrA binders were evaluated by electrophoretic mobility shift assays (EMSAs). A 300-bp promoter region of porGDAB operon was amplified by PCR and used as target sequence of HsrA in all EMSA experiments^{14 (link)}. Recombinant HsrA protein (6 μM) was mixed with 120 ng of target promoter in a 20 μL reaction volume containing 10 mM bis-Tris (pH 7.5), 40 mM KCl, 100 mg/L BSA, 1 mM DTT, and 5% glycerol. During the inhibition assays, putative inhibitors were added to final concentrations of 2, 1, 0.5 and 0.1 mM to the mixtures of protein and DNA. Binding assays with DMSO instead of inhibitors were included as vehicle controls. To ensure the specificity of EMSAs, a 150-bp DNA fragment corresponding to the coding region of gene pkn22 (alr2502) from Anabaena sp. PCC 7120 was included as non-specific competitor DNA in all assays. Mixtures were incubated at room temperature for 20 min and subsequently separated on a 6% non-denaturing polyacrylamide gel in running buffer (25 mM Tris, 190 mM glycine) at 90 V. The gel was stained with SYBR Safe DNA gel stain (Invitrogen) and processed with a Gel Doc 2000 Image Analyzer (Bio-Rad).

Recombinant Anabaena sp. PCC 7120 FurA protein was expressed in Escherichia coli BL21 (DE3) and purified according to previously described methods (43 ). The promoter regions of each gene of interest were obtained by PCR using the primers described in Supplementary Table S1. Electrophoretic mobility shift assays (EMSA) were performed as described previously (36 (link)). Briefly, 120–140 ng of each DNA fragment were mixed with recombinant FurA protein at concentrations of 300, 500 and 700 nM in a 20-µl reaction volume containing 10 mM bis–Tris (pH 7.5), 40 mM potassium chloride, 100 µg/ml bovine serum albumin, 1 mM DTT, 100 µM manganese chloride and 5% glycerol. In some experiments, EDTA was added to a final concentration of 200 μM. To ensure the specificity of EMSA, the promoter region of Anabaena sp. nifJ (alr1911) gene was included as non-specific competitor DNA in all assays (31 (link)). Mixtures were incubated at room temperature for 20 min, and subsequently separated on 4% non-denaturing polyacrylamide gels in running buffer (25 mM Tris, 190 mM glycine) at 90 V. Gels were stained with SYBR® Safe DNA gel stain (Invitrogen) and processed with a Gel Doc 2000 Image Analyzer (Bio-Rad).