Hi! Personnally, i've worked with S9 strain but...it should be similar...S9 is just more prone to produce PM. At 25% of salt, you dont need any antibiotic, nothing grows other than Halophile. A high amount of 850 W flood light is what's important for purple membrane induction instead of red membrane. I remember a paper that was about downsizing PM with sonication...but it was 10 years ago...For instance:
http://www2.thphy.uni-duesseldorf.de/~hlowen/doc/op/op0081.pdf
Bacterial strains and cell culture conditions
Halobacterium halobium R1 was obtained (ATCC29341; American Type Culture Collection, Manassas, VA). Oxoid peptone medium, from Oesterhelt and Stoeckenlius's formula, contained the following components per liter: NaCl, 250 g; MgSO4, 9.8 g; KCl, 2.0 g; Tri-sodium citrate·2H2O, 3.0 g; Oxoid bacteriological peptone (Cat. No. LP0037, Oxoid, Basingstoke, Hampshire, UK), 10 g. Cell cultures were placed under 850 W flood light (∼50 μmol s−1 m−2), while shaken for 90–100 h at 37°C, 200 rpm, followed by static cultivation for two days. Cell growth was monitored by the ultraviolet/visible spectrometer (Nanodrop 2000c with special cuvette for cell density measurement, Thermo Scientific, Waltham, MA) at 660 nm.
Purple membrane purification
Cells from 1 L culture were harvested by centrifugation (15,000 g) for 15 min. Cell pastes were then resuspended in 25 mL basal salt (medium without peptone) and 50 μL DNase I (2000 U/mL, New England Biolabs, Ipswich, MA) and dialyzed against 2 L of deionized water overnight. The clean red dialysate was centrifuged at 50,000 g for 40 min and resuspended in 35 mL deionized water. After homogenization by a tissue grinder with a tight-fitting Teflon pestle, solution was centrifuged at 50,000 g. This step was repeated two times until the supernatant was nearly colorless.
The purple pellet was then resuspended in 1 mL deionized water. A linear 30–50% (w/w) with a 60% cushion sucrose density gradient centrifugation at 100,000 g, 22 h was used to separate the purple membrane and the red membrane at 15°C (see Fig. S1 in the Supporting Material). The isolated purple band was dialyzed against 1 L deionized water for 3 h with one water change. The dialysate was centrifuged at 14,000g for 30 min, and finally resuspended in 250 μL deionized water and stored at −20°C.
Good luck!
Dan
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