Shewanella oneidensis outer membrane and outer membrane vesicles

This project compares the outer membrane proteome to the proteome of outer membrane vesicles of the bacterium Shewanella onedensis. S. oneidensis outer membrane was purified via the sarkosyl method described in (Brown, 2010). A 50 mL overnight culture of cells were harvested by centrifugation at 10,000× g for 10 min. cell pellet suspended in 20 mL of 20 mM ice-cold sodium phosphate (pH 7.5) and passed four times through a French Press (12000 lb/in2). The lysate was centrifuged at 5,000× g for 30 min to remove unbroken cells. The remaining supernatant was centrifuged at 45,000 × g for 1 h to pellet membranes. Crude membranes were suspended in 20 mL 0.5% Sarkosyl in 20 mM sodium phosphate and shaken horizontally at 200 rpm for 30 min at room temperature. The crude membrane sample was centrifuged at 45,000 × g for 1 h to pellet the OM. The pellet of OM was washed in ice-cold sodium phosphate and recentrifuged. The cells were pelleted by centrifugation at 5000 x g for 20 min at 4°C, and the supernatant was filtered through 0.45 μm pore size filters to remove remaining bacterial cells. Vesicles were obtained by centrifugation at 38,400 x g for 1 h at 4°C in an Avanti J-20XP centrifuge (Beckman Coulter, Inc). Pelleted vesicles were resuspended in 25 ml of 50 mM HEPES (pH 6.8) and filtered through 0.45 μm pore size filters. Vesicles were again pelleted as described above and finally resuspended in 50 mM HEPES, pH 6.8. Extracellular DNA, flagella, and pili can all be co-purified. Protocol was adapted from (Perez-Cruz, 2013).