pHIL-D2- xynBS27

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	The recombinant pHIL-D2- xynBS27 plasmid was linearized with SacI and then transformed into P. pastoris GS115 and SMD1168 competent cells by electroporation using a Gene Pulser electroporator (Bio-Rad, Hercules, CA, USA).

Protocol tips
The recombinant pHIL-D2- xynBS27 plasmid was linearized with SacI and then transformed into P. pastoris GS115 and SMD1168 competent cells by electroporation using a Gene Pulser electroporator (Bio-Rad, Hercules, CA, USA).

Publication protocol

"A xylanase gene xynBS27 (accession number EU660497) from Streptomyces sp. S27 was synthesized by Eurofins® according to the codon usage for P. pastoris. The synthetic gene encoding the XynBS27 was digested with EcoRI and subcloned into pHIL-D2 vector under control of alcohol oxidase 1 (AOX1) promoter. The recombinant pHIL-D2- xynBS27 plasmid was linearized with SacI and then transformed into P. pastoris GS115 and SMD1168 competent cells by electroporation using a Gene Pulser electroporator (Bio-Rad, Hercules, CA, USA).

Transformants cells were initially selected by the ability to grow on Minimal Dextrose (MD) agar plates [1.34% (w/v) Yeast Nitrogen Base (YNB) (Invitrogen, Carlsbad, CA, USA), 4 x 10−5% (w/v) biotin and 1.0% (w/v) dextrose] without histidine. The integration of expression cassette into the genome of pHIL-D2- xynBS27 strains was verified by PCR using AOX5 (5’-GACTGGTTCCAATTGACAAGC-3’) and AOX3 (5’-GCAAATGGCATTCTGACATCC-3’) primers in accordance with the instructions of the P. pastoris expression kit (Invitrogen, Carlsbad, CA, USA).

Selected positive colonies were cultured in 1-L Erlenmeyer flasks containing 100 mL of BMGY-U medium at 28 °C under shaking at 200 rpm, until optical density at 600 nm reached 5–6. The cells were collected by centrifugation (12.000 x g for 15 min) and transferred to 1-L Erlenmeyer flasks containing 100 mL of BMMY-U medium, followed by incubation at 28 °C under shaking at 200 rpm for 7 days. To maintain induction, methanol was added every day to a final concentration in the range of 1 to 4% (v/v). After 7 days of induction, the cells were harvested by centrifugation at 12.000 x g for 15 min, and supernatant was used as source of xylanase."

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