Editing DSMZ Cells (section)

=== Chinese Method ===

''T. maritima'' strain MSB8 (JCM10099), a gift from Institute of Microbiology, Chinese Academy of Sciences, was activated and cultured at 80°C in modified medium containing (per liter of distilled water) 10.0 g starch, 5.0 g yeast extract, 10.0 g tryptone, 27.0 g NaCl, 0.5 g KH<sub>2</sub>PO<sub>4</sub>, 0.5 g K<sub>2</sub>HPO<sub>4</sub>, 0.5 g MgCl<sub>2</sub>·6H<sub>2</sub>O, 0.33 g KCl, 1.0 g NH<sub>4</sub>Cl, 0.08 g CaCl<sub>2</sub>·2H<sub>2</sub>O, 0.5 g L-cysteine, 0.5 g Na<sub>2</sub>S·9H<sub>2</sub>O, 1.0 mg resazurin and 15.0 ml of trace minerals solution containing (per liter distilled water) 1.5 g nitrilotriacetic acid, 3.0 g MgSO<sub>4</sub>·7H<sub>2</sub>O, 0.5 g MnSO<sub>4</sub>, 1.0 g NaCl, 0.1 g FeSO<sub>4</sub>·7H<sub>2</sub>O, 0.1 g CoSO<sub>4</sub>·7H<sub>2</sub>0, 0.1 g ZnSO<sub>4</sub>·7H<sub>2</sub>O, 0.01 g CuSO<sub>4</sub>·5H<sub>2</sub>O, 0.01 g AlK(SO<sub>4</sub>)<sub>2</sub>, 0.01 g H<sub>3</sub>BO<sub>3</sub> and 0.01 g Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O. The medium was boiled under a stream of oxygen-free N<sub>2</sub> gas and cooled to 80°C. Two milliliters of the activated bacteria were inoculated to 100 ml of medium and then cultured at 60, 70, 80 or 90°C. To monitor the growth rates, the cultured bacteria were withdrawn at time intervals, every 2 hours, and their O.D.<sub>600 nm</sub> was measured. The growth curves of the bacteria were generated by fitting the data to the regression equation A = A<sub>0</sub>+a/(1+Exp(T<sub>1/2</sub>−''t'')/b), in which A is the O.D. value at 600 nm, ''t'' is the culture time, A<sub>0</sub> is the O.D.<sub>600 nm</sub> at lag phase and T<sub>1/2</sub> is the time when the cultures reach the middle of log phase. The “a” constant is the maximum change in the O.D.<sub>600 nm</sub> between A<sub>max</sub> and A<sub>0</sub>, while “b” is the time between t<sub>lag end</sub> and t<sub>log end</sub>. When the bacteria reached T<sub>1/2</sub>, the cells were considered at optimal conditions; the bacteria were harvested by centrifuge at 6,000 g for 15 min at 4°C followed by a wash with ultrapure water. The bacterial cultures at each temperature were prepared in triplicate.


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