Escherichia coli strain NEB10β was used for all cloning and plasmid propagation. NEB10β was cultivated at 37 °C in lysogeny broth (LB) supplemented with 50 μg/mL of ampicillin. Saccharomyces cerevisiae strain BY4741 (Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) was routinely cultivated at 30 °C in yeast extracted peptone dextrose (YPD) medium, or yeast synthetic complete medium (YSC). YPD is composed of 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose. YSC dropout medium is composed of 6.7 g/L yeast nitrogen base, 20 g/L glucose or galactose, and CSM dropout supplement (MP Biomedicals, Solon, OH).
Plasmids were transformed to E. coli by electroporation in 2 mm cuvettes at 2.5 kV and 25 μF using a Gene Pulser Xcell (Bio-Rad). Plasmids were subsequently isolated from E. coli using the GeneJET Plasmid Miniprep kit (Thermo). All S. cerevisiae integrations were done by the EZ Yeast Transformation II Kit (Zymo) according to manufacturer's instruction. Correct construct integrations in biological triplicate in S. cerevisiae were confirmed through colony PCR and subsequent Sanger sequencing.
Plasmids were transformed to E. coli by electroporation in 2 mm cuvettes at 2.5 kV and 25 μF using a Gene Pulser Xcell (Bio-Rad). Plasmids were subsequently isolated from E. coli using the GeneJET Plasmid Miniprep kit (Thermo). All S. cerevisiae integrations were done by the EZ Yeast Transformation II Kit (Zymo) according to manufacturer's instruction. Correct construct integrations in biological triplicate in S. cerevisiae were confirmed through colony PCR and subsequent Sanger sequencing.