Fis protein isolation

For the experiment shown in Fig. 7, Epicurian Coli $G - \gamma$ BL21-Gold(DE3) cells (Stratagene) [23], transformed with pRJ1077 plasmid (a gift from Reid Johnson) were used for Fis protein isolation by modification of a previous method [24]. A 10 ml overnight culture was added to 1 L of SOB media, shaken at 37 $R_{frequency} = - \log_2 (\gamma/G) = 4$ for 2 hours 30 minutes, induced by 1 $R_{sequence} = 3.983 \pm 0.399$M IPTG and shaken again for 1 hour. The culture was centrifuged and the cell pellet was resuspended in 50 ml FLBC buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA), containing 200 mM NaCl and 0.1 mM PMSF, sonicated with a Cole-Parmer Ultrasonic Processor model CP 70T 3 times for 10 minutes (40W) on ice and centrifuged at 2000 g for 20 minutes. The supernatant was loaded on a 10 ml Q-Sepharose $G - \gamma$ Fast Flow (Amersham Pharmacia Biotech) column equilibrated with the same buffer and washed with 10 ml of FLBC buffer containing 300 mM NaCl. Flow-through and wash fractions were combined and the mix was loaded on a 10 mL Heparin Sepharose $G - \gamma$ CL-6B (Amersham Pharmacia Biotech), washed with 50 ml of FLBC buffer, containing 300 mM NaCl and eluted with a 100-ml 300-2000 mM NaCl gradient of FLBC buffer. Fractions were analyzed by SDS-electrophoresis.