Adenine

pYC2/CT plasmids were transformed into S. cerevisiae BJ5465 fex:GSHU fex2Δ using the lithium-acetate/PEG method (Gietz, 2014 (link)). Transformants were selected on SD-URA plates (20 g/L D-glucose, 6.7 g/L yeast nitrogen base (Becton, Dickinson & Co., Sparks, MD, USA), 5 g/L casamino acids (Becton, Dickinson & Co., Sparks, MD, USA), 40 mg/L Tryptophan, 40 mg/L Adenine, 16.25 g/L sodium citrate dihydrate, 4.2 g/L citric acid monohydrate, 20 g/L agar (Becton, Dickinson & Co., Sparks, MD, USA)). Genes were expressed essentially as described in Drew et al., with the following modifications (Drew et al., 2008 (link)). Briefly, individual colonies were used to inoculate 3 mL overnight cultures in SD-URA medium (20 g/L D-glucose, 6.7 g/L yeast nitrogen base (Becton, Dickinson & Co., Sparks, MD, USA), 5 g/L casamino acids (Becton, Dickinson & Co., Sparks, MD, USA), 40 mg/L Tryptophan, 40 mg/L Adenine, 16.25 g/L sodium citrate dihydrate, 4.2 g/L citric acid monohydrate) and grown at 30 °C, 225 rpm, for ∼16 h. The overnight cultures were used to seed an expression culture in SR-URA medium (same composition as SD-URA, except with 20 g/L raffinose instead of glucose) to a starting OD₆₀₀ of 0.12. The culture was grown at 30 °C, 225 rpm for 6–7 h or until OD₆₀₀ ∼0.6–1, when gene expression was induced with 2% w/v galactose for 22–24 h (30 °C, 225 rpm).