The mixture was suspended in fresh media, serially diluted and plated on soybean mannitol agar click here plates containing 10 mM MgCl2. After 18 h incubation at 30 °C, the plates were overlaid with 500 μg nalidixic acid to inhibit E. coli growth. Depending on the plasmid marker, either apramycin (1 mg) or thiostrepton (0.5 mg) were overlaid to select for exconjugants. Exconjugants usually appeared on the 5th day after drug overlay. Individual
exconjugants were taken forward for further generations. Proteins separated by SDS-PAGE were transferred to nitrocellulose membrane by semi-dry electroblotting. After electrophoresis, the gel was soaked in ice-chilled transfer buffer for 20 min. On the anode plate of the apparatus, three layers of Whatman-3 filter papers were placed.
Nitrocellulose membrane soaked in Towbin transfer buffer was placed on the filter paper wad. Then the gel was placed on the membrane, followed by a wad of four Whatman-3 papers soaked with transfer buffer. The cathode plate and safety cover CB-839 purchase were placed in position and blotting was done at 250 mA for 2 h. rDnrO protein 50 μg was incubated with 20 ng of DNR for 30 min at room temperature and was centrifuged in a 10-kDa membrane centrifugal concentrator (as shown by Prasad et al., 2003) for 20 min at 6000 g at 4 °C. The retentate was suspended in 10 mM Tris pH 7.5, and analyzed using a spectrophotometer at 590 nm for the presence of DNR complexed with DnrO. Streptomyces lividans TK24 carrying
pIJ8660/dnrNO plasmid was grown in nitrate-defined yeast extract medium for 36 h at 30 °C. It was further subcultured in nitrate-defined yeast extract medium with 2 ng mL−1 DNR and incubated for 48 h at 30 °C. Mycelium was quickly washed with phosphate-buffered saline (PBS) and the expression of EGFP was visualized using a confocal microscope (Leica) at 60 × magnification. The 37-bp DnrO-binding sequence was used to construct a 3D model using ‘model it server’ (http://hydra.icgeb.trieste.it/dna/model_it.html). ADAMTS5 The structure of DNR was downloaded from protein data bank (http://www.rcsb.org/) as a pdb file. autodock (http://autodock.scripps.edu/) was used to dock the DNR molecule to the 37-bp DNA to find preferred binding sequences. DNA-binding ELISA (Renard et al., 2001) was performed using Sigma screen streptavidin-coated high-capacity microplates. Biotinylated 511-bp (intergenic region of dnrN and dnrO genes carrying the 37-bp DnrO-binding site) DNA (10 ng) in TE was immobilized to ELISA plates at room temperature. Wells were washed thoroughly with PBS containing 0.1% Tween-20. Different amounts of rDnrO were added to test the binding capacity to immobilized DNA and were incubated for 1 h at 30 °C. Unbound DnrO was washed with PBS containing 1% Tween-20, following which, primary (anti-DnrO) antibody was added.