Salmon sperm dna

DNA fragments used for the electrophoretic mobility shift assay (EMSA) were PCR amplified using Cy5-labeled primers to perform a non-radioactive EMSA. DNA fragments used were the upstream region of acrD (246 bp), and as controls, the upstream regions of acrAB (205 bp) and tolC (291 bp). Approximately 0.16 pmol of Cy5-labeled DNA was mixed with increasing concentrations of His-tagged BaeR protein in a binding buffer reaction (50 mM Tris–HCl, pH 7.5; 1 mM DTT; 500 mM MgCl₂; 100 mM EDTA; 10 mM NaCl; 5% glycerol). To decrease unspecific binding, 500 ng competitor DNA (Salmon sperm DNA, AppliChem) was added to the reaction. Incubation was done at room temperature for 30 min. The total reaction was run on a native 4% polyacrylamide gel in 0.5x Tris-borate-EDTA (TBE) buffer at constant 25 mA. After electrophoresis, fluorescence signals of the labeled DNA were visualized using a FLA-3000 phosphorimager (Raytest, Straubenhardt, Germany).

DNA fragments used for the electrophoretic mobility shift assay (EMSA) were PCR amplified using Cy5-labeled primers to perform a non-radioactive EMSA. DNA fragments used were the upstream region of mdtABC (290 bp), mdtUVW (276 bp) and as control, a fragment of the tolC gene (248 bp). Approximately 0.3 pmol of Cy5-labeled DNA was mixed with increasing concentrations of His-tagged BaeR or CpxR protein in a binding buffer reaction (10 mM Tris–HCl, pH 7.5; 50 mM KCl; 5 mM MgCl₂; 1 mM DTT; 2.5% glycerol). To decrease unspecific binding, 50 ng competitor DNA (Salmon sperm DNA, AppliChem) was added to the reaction. Incubation was done at room temperature for 30 min. The total reaction was run on a native 4% polyacrylamide gel in 0.5× Tris-borate-EDTA (TBE) buffer at constant 25 mA. After electrophoresis, fluorescence signals of the labeled DNA were visualized using a FLA-3000 phosphorimager (Raytest, Straubenhardt, Germany).

M. pulcherrima was transformed with a modified protocol from [11 (link)]. Overnight cultures were diluted to an OD₆₀₀ = 0.3 and grown until they reach OD₆₀₀ = 0.8–1. 1 mL of culture per transformation was pelleted, washed once with PBS (Oxoid, Leicester, UK), then were resuspended in 260 µL of transformation mix 4 µg of linearized DNA, 100 µL 10 × tris-EDTA (Fisher Scientific, Loughborough, UK), 100 µL 1 M lithium acetate (Sigma, St. Louis, MO, USA) pH 7.4, 40 µL 5 mg/mL salmon sperm DNA (AppliChem, Darmstadt, Germany) previously boiled, 20 µL 1 M DTT (Sigma, St. Louis, MO, USA), then 800 µL of polietinelglicol (PEG, Sigma, St. Louis, MO, USA) 3350 was added. Transformations were incubated at 25 °C overnight. The cells were heat shocked at 40 °C for 5 min then placed on ice for 1 min. After centrifugation for 5 min at 1100 g, the supernatant was removed and the cells were resuspended in SMB, then incubated for 2 h at 25 °C, 200 rpm, then plated on to MEA containing 50 μg/mL Nat and placed in a static incubator at 25 °C for 2–3 days until colonies appeared.