Molecular Cloning and Transformation Protocols in Yeast

All restriction enzymes were purchased from FastDigest Thermo Scientific and all of the digestions were performed according to standard protocols. The PCR reactions were set up using recommended conditions and Phusion high-fidelity DNA polymerase (Thermo Scientific). The ligation reactions were performed for 10 min at room temperature using T4 DNA Ligase (Thermo Scientific). The gel extractions were performed using the Gel Out extraction kit purchased from A&A Biotechnology (Poland). The E. coli minipreps were performed using the Plasmid Mini Kit (A&A Biotechnology). Transformation of E. coli strains was performed using standard chemical protocols.
Transformation was performed by the lithium acetate method and transformants were plated out on selective media without uracil as described before (Mirończuk et al., 2016 (link)). They were analyzed for proper integration by gDNA extraction and PCR amplification. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland).