Yeast Genetic Manipulation by Co-transformation

All mutagenesis experiments were carried out by co-transformation of 10 µL of the pMZ374 vector series with 20 μL of the respective PCR product being used as the HR donor DNA for the target locus, as described in a previous report [17 (link)]. Briefly, strains were grown in 5 mL of YM medium at 30 °C with shaking at 180 rpm until the culture reached an optical density (OD) at 600 nm of 0.5; cells then were co-transformed by electroporation using a Gene Pulser Xcell II (Bio-Rad) and standard methodologies. Transformants were selected using EMM+Leu plate, then screened by colony PCR and DNA sequencing.