Cupriavidus metallidurans CH34 [29], [29], Escherichia coli JM109 (pTP6) [26] and Cupriavidus necator JMP134 (pJP4) [30] were grown in Luria Bertani (LB) medium (contained per l: 10 g tryptone, 5 g yeast extract and 10 g NaCl) [31] in absence or presence of Hg2+.
ZUC99 pATX210) also exhibited efficient conversion in fermentor experiments with 1 l medium containing l-arabinose and glycerol.
The transformed bacteria were cultured at 30 °C in 100 mL of LB medium containing the appropriate antibiotics overnight.
A two-milliliter culture of M90T-Sm pRR008 was grown overnight, then subcultured at a ratio of 1 100 in 50 mL of medium containing 1% L-arabinose the next morning.
The cells were split at 1 : 10 and cultured in the medium containing 500 μg ml−1 of the G418 (Gibco) for 21 days.
After removing the culture medium, the wells were washed with PBS and incubated with freshly prepared solutions of ALA or ALA peptide derivatives 1– 10: 0.1 ml of serum-free medium containing varying prodrug concentrations was added to a designated series of wells.
For the hypo-osmotic stress experiment (Fig. 6e,f), cells were grown overnight in SD medium containing 1 M sorbitol, diluted in the morning in the same medium to OD600 0.05, and grown for at least 4 hours before the start of the experiment.
The cells were pelleted as before and resuspended in growth medium (Clonetics MEGM complete medium, Lonza, Basel, Switzerland) and seeded at a density of 2.5 × 106 cells per well in a six-well plate pre-incubated with 1 mL/well growth medium containing 2 mg/mL fetuin and grown overnight.
Transformed H. salinarum cells were grown in complex medium containing 1 mg/mL mevinolin as described previously (7).
The transformed H. salinarum cells were grown to early stationary phase in complex medium containing 1 mg/mL mevinolin as described previously (7).
