3β-HSD plasmid and library construction

The 3β-HSD ORF was synthesized as double-stranded DNA (Integrated DNA Technologies, Inc., Coralville, IA) and amplified using primers oJF325 and oJF326 using KAPA HiFi under standard PCR conditions and digested with BsmBI to create plasmid pJF57. 3β-HSD expression plasmids (pJF76 through pJF87) were generated by digesting plasmid pJF57 along with corresponding plasmids from the Yeast Cloning Toolkit (Lee et al., 2015) with BsaI and assembled using the Golden Gate Assembly method (Engler et al., 2008). The 3β-HSD sequence was randomized by error-prone PCR using a Genemorph II kit from Agilent Technologies. An aliquot containing 100 ng of target DNA was mixed with 5 μL of 10X Mutazyme buffer, 1 μL of 40 mM dNTPS, 1.5 μL of 20 μM forward and reverse primer containing 90-bp overlap with the 3β-HSD expression plasmids and 1 μL of Mutazyme polymerase in 50 μL. The reaction mixture was subject to 30 cycles with Tm of 60°C and extension time of 1 min. Vector backbone was amplified using KAPA HiFi polymerase with oJF387 and oJF389 (pPAB1) or oJF387 and oJF389 (pPOP6) with Tm of 65°C and extension time of 350 s. PCR products were isolated by 1.5% agarose gel electrophoresis and assembled using the Gibson method (Gibson et al., 2009). Assemblies were pooled, washed by ethanol precipitation, and resuspended in 50 μL of dH₂O, which was drop dialyzed (Millipore) and electroporated into E. cloni supreme cells (Lucigen). Sanger sequencing of 16 colonies showed a mutation rate of 0–4 mutations/kb. The library was expanded in culture and maxiprepped (Qiagen) to 500 μg/μL aliquots. 16 μg of library was drop dialyzed and electrotransformed into yeast strain PyE1.