Editing DSMZ Cells (section)

=== Method 3 ===

T. maritima strain MSB8 and M. jannaschii strain DSM2661&nbsp; were&nbsp; grown&nbsp; at&nbsp; 80°C&nbsp; on&nbsp; medium&nbsp; (BSMII)&nbsp; containing:&nbsp; 40&nbsp; g/liter&nbsp; sea salts, 5 g/liter tryptone, 3 g/liter yeast extract, 3 g/liter maltose, 3 g/liter piperazine-1,4-bis(2-ethanesulfonic&nbsp; acid)&nbsp; (PIPES),&nbsp; 0.5&nbsp; mg/liter&nbsp; NaSeO3·5H2O,&nbsp; 0.5 mg/liter NiCl2·6H2O, 0.25 g/liter NH4Cl, 10 mg/liter Fe(NH4)2(SO4)2·6H2O, 1.25&nbsp; g/liter&nbsp; sodium&nbsp; acetate&nbsp; anhydrate,&nbsp; 1&nbsp; ml/liter&nbsp; 1%&nbsp; Resazurin,&nbsp; 10&nbsp; ml/liter Wolfe’s trace elements, and 10 ml/liter Wolfe’s trace vitamins solution (all from Sigma&nbsp; Aldrich;&nbsp; components&nbsp; of&nbsp; Wolfe’s&nbsp; vitamin&nbsp; and&nbsp; mineral&nbsp; solutions&nbsp; can&nbsp; be found in ATCC medium no. 1343). The medium was adjusted to pH 7.0, autoclaved for sterility, and stored at 4°C until use. For H2 inhibition experiments, 400&nbsp; ml&nbsp; of&nbsp; medium&nbsp; was&nbsp; dispensed&nbsp; into&nbsp; a&nbsp; 1-liter&nbsp; glass&nbsp; jar&nbsp; sealed&nbsp; with&nbsp; a&nbsp; rubber septum&nbsp; (Corning,&nbsp; Corning,&nbsp; NY)&nbsp; and&nbsp; heated&nbsp; to&nbsp; 80°C.&nbsp; The&nbsp; medium&nbsp; was&nbsp; then sparged&nbsp; through&nbsp; a&nbsp; syringe&nbsp; needle&nbsp; with&nbsp; the&nbsp; appropriate&nbsp; gas&nbsp; (100%&nbsp; N2 or&nbsp; 80%<br/>H2–20% CO2) for 1 minute and reduced with 4 ml of a 10% sodium sulfide–10% L-cysteine HCl solution (pH 8.0) (Sigma-Aldrich). A 1% inoculum of log-phase cells was used. Culture bottles were agitated at 100 rpm in an oil bath at 80°C