Top10 chemically competent escherichia coli cells

TOP10 chemically competent Escherichia coli cells (Invitrogen, Carlsbad, CA) were used for all plasmid maintenance and cloning. Wild-type S. stipitis strain CBS 6054 (ATCC 58785) was used for all yeast experiments. E. coli were grown in solid or liquid LB (Luria-Bertani) medium containing 100 µg/mL ampicillin overnight. S. stipitis was grown overnight at 30°C in liquid YPD (1% yeast extract, 2% peptone, and 2% dextrose) or on solid YPD (2% agar) with or without one of the following antibiotics: 100 µg/mL Zeocin (Invitrogen, Carlsbad, CA), 200 µg/mL hygromycin B (RPI, Mt. Prospect, IL), 10–100 µg/mL Nourseothricin (RPI), or 200 µg/mL G-418 (RPI). For some ura3 deletion experiments, S. stipitis was grown on synthetic minimal medium without uracil (6.7 g/L Difco yeast nitrogen base without amino acids, 770 mg/L, Formedium CSM drop-out mix without uracil, 2% glucose, and 2% agar). For other experiments, this minimal medium was supplemented with 100 µg/mL of uracil.

Amplicons were ligated into pcDNA3.1-Topo Directional Cloning Vector (Invitrogen, Life Technologies, Carlsbad, CA), transformed into Top10 chemically competent Escherichia coli cells (Invitrogen, Life Technologies, Carlsbad, CA) as per the manufacturer’s instructions, and cultured on Luria-Bertani agar supplemented with 100 μg/mL carbenicillin (Sigma-Aldrich, St. Louis, MO). Plasmid purification was performed using the QIAprep spin miniprep, or the QIAfilter plasmid midi kits (Qiagen, Valencia, CA), as per the manufacturer’s instructions. Plasmids were sequenced as described above in order to ensure the clone contained no nonsynonymous mutations relative to the SGA-derived env sequence.