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	After digestion with AscI and BspHI the transformation of the P. pastoris wild type strain X-33 was performed by electroporation. The repressible promoter cassette should interact with the genomic DNA in the cell and replace the respective promoter of the gene of interest (Figure 4B) [15].

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After digestion with AscI and BspHI the transformation of the P. pastoris wild type strain X-33 was performed by electroporation. The repressible promoter cassette should interact with the genomic DNA in the cell and replace the respective promoter of the gene of interest (Figure 4B) [15].

Publication protocol

P. pastoris genes ERO1 (PAS_chr1-1_0011) and PDI1 (PP7435_Chr4-0183), and 500 bp of their respective promoter regions (starting −200 bp upstream from the ATG) were amplified by PCR from X-33 genomic DNA (primers are listed in Table 2). The expression cassette was generated by flanking the resistance cassette Zeocin 5’ with ~ 500 bp of native promoter sequence of the gene of interest, and 3’ by the repressible promoter, which was followed by the P. pastoris gene of interest (Figure 4A). We combined the repressible promoter PTHR1 with ERO1 and PTHI11 with PDI1. After digestion with AscI and BspHI the transformation of the P. pastoris wild type strain X-33 was performed by electroporation. The repressible promoter cassette should interact with the genomic DNA in the cell and replace the respective promoter of the gene of interest (Figure 4B) [15].

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