Saccharomyces cerevisiae CEN.PK102-5B (MATa ura3-52 his3Δ1 leu2-3/112 MAL2-8cSUC2) strain was obtained from Verena Siewers (Chalmers University). Yeast transformants were selected on synthetic complete (SC) drop-out media lacking the amino acids matching the auxotrophic markers on the plasmids used. These SC plates were made from premixed drop-out powders from Sigma-Aldrich. When yeast was grown in liquid media, it was either in SC, Delft, or standard yeast peptone dextrose (YPD) media. Delft contained (L−1): 7.5 g (NH4)2SO4, 14.4 g KH2PO4, 0.5 g MgSO4·7H2O, 22 g dextrose, 2 mL trace metals solution, and 1 mL vitamins. The pH of Delft medium was adjusted to 6 prior to autoclavation. Vitamin solution was added to Delft medium after autoclavation. Vitamin solution was added after autoclavation. The trace metals solution contained (L−1): 4.5 g CaCl2·2H2O, 4.5 g ZnSO4·7H2O, 3 g FeSO4·7H2O, 1 g H3BO3, 1 g MnCl2·4H2O, 0.4 g Na2MoO4·2H2O, 0.3 g CoCl2·6H2O, 0.1 g CuSO4·5H2O, 0.1 g KI, 15 g EDTA. The trace metals solution was prepared by dissolving all the components except EDTA in 900 mL ultra-pure water at pH 6. The solution was then gently heated and EDTA was added. In the end, the pH was adjusted to 4, and the solution volume was adjusted to 1 L and autoclaved (121 °C in 20 min). This solution was stored at + 4 °C. The vitamin solution had (L−1): 50 mg biotin, 200 mg p-aminobenzoic acid, 1 g nicotinic acid, 1 g Ca-pantothenate, 1 g pyridoxine-HCl, 1 g thiamine-HCl, 25 g myo-inositol. Biotin was dissolved in 20 mL 0.1 M NaOH and 900 mL water is added. pH was adjusted to 6.5 with HCl and the rest of the vitamins were added. pH was re-adjusted to 6.5 just before and after adding m-inositol. The final volume was adjusted to 1 L and sterile-filtered before storage at + 4 °C.
All standard cloning was carried out using Escherichia coli strain DH5α, which was grown in standard Luria–Bertani (LB) medium containing 100 μg mL−1 ampicillin. For the cloning of plasmid carrying the ccdB gene and chloramphenicol cassette, E. coli ccdB strain was used as a host strain and transformants were selected on LB medium containing 100 μg mL−1 ampicillin and 25 μg mL−1 chloramphenicol.
All standard cloning was carried out using Escherichia coli strain DH5α, which was grown in standard Luria–Bertani (LB) medium containing 100 μg mL−1 ampicillin. For the cloning of plasmid carrying the ccdB gene and chloramphenicol cassette, E. coli ccdB strain was used as a host strain and transformants were selected on LB medium containing 100 μg mL−1 ampicillin and 25 μg mL−1 chloramphenicol.
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