GSM8247864: ɑBAK1, naïve; Saccharomyces cerevisiae; OTHER
Yeast were lysed with 125 U/ml Zymolase at 37 ºC for 5 h, and DNA was harvested (Zymoprep kit from Zymo Research). Genomic DNA was digested with 2 U/µl Exonuclease I and 0.25 U/µl Lambda exonuclease (New England Biolabs) for 90 min at 30 ºC, and plasmid DNA purified with a QIAquick kit (Qiagen). In some instances, gene sequences were diversified by overlapping PCR for site-directed mutagenesis libaries as described in Procko et al. J Mol Bio 2013. In other instances, gene sequences were commercially synthesized. Genes were prepared with 5' and 3' overhangs >20 bp to enable homologous recombination with the plasmid backbone (pETCON) for yeast expression and surface display via fusion to Aga2p. DNA amplicon sequencing