Gel out extraction kit

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 protocols¹⁷ .
For transformation of Yarrowia lipolytica only strains with auxotrophy for uracil were used.
Transformation was performed according to the lithium acetate method^{24 (link)} and transformants were plated out on selective media without uracil. They were analyzed for proper integration by gDNA extraction and PCR amplification with two primer pairs. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland).

The vector carrying hp0231m was constructed by a two-step PCR method. Briefly, primers HP231_BamL and HP231_katR were used to amplify the DNA region encoding promoter region of the hp0231 gene with a native signal sequence (SS) from the chromosome of H. pylori 26695. The catalytic domain of the hp0231 gene, including the active motif CXXC, was amplified by HP231_katL and HP231_XhoR primers, also from the chromosome of H. pylori 26695. The HP231_katL and HP231_katR primers contained 5′ leader nucleotide sequences complementary to each other. Each PCR product was purified by a Gel-Out extraction kit (A&A Biotechnology). Next a mixture of two purified products (in equal amounts) was used as a template in a single PCR reaction, using the primers HP231_BamL and HP231_XhoR. Subsequently, the resulting PCR product was purified and cloned into pGEM-T Easy, generating pUWM568. Finally, using BamHI and XhoI restriction enzymes, the 1.5 kb DNA region encoding hp0231m was transferred into pHel2 and pHel3, generating pUWM575 and pUWM574, respectively. Correct construction of the resulting plasmids was verified by sequencing. Production of a proper protein was confirmed by Western-blot using anti-HP0231 serum.

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).