DNA Gyrase Supercoiling Assay

DNA gyrase supercoiling assays, using gel electrophoresis, were carried out based on published procedures (17 (link)) as follows. Reactions (30 μl) contained 1 μg relaxed plasmid DNA, in 35 mM Tris–HCl (pH 7.5), 24 mM KCl, 4 mM MgCl₂, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol, 0.1 mg/ml albumin (John Innes Enterprises) and were incubated at 37°C for 30 min. Samples were analysed either using microplate assays (below) or by electrophoresis on 1% agarose gels; results from gel assays were quantitated using the intensity of the ethidium fluorescence of the supercoiled DNA band using a Syngene GelDoc system. Where indicated, ciprofloxacin and novobiocin were also added to assays. Topo I, topo II and topo IV assays were carried out according to the manufacturer's instructions (Promega, Topogen and John Innes Enterprises Ltd) using 1 μg supercoiled plasmid DNA as substrate.