pKS2-ST::CBHII

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	The pKS2-ST::CBHII vector was used to transform competent cells of W. anomalus 54-A by electroporation, using the Gene Pulser electroporation system Xcell™ (Bio-Rad Laboratories) at 1400 V and 200 Ω. The W. anomalus 54-A clones were selected in YPD medium supplemented with 500 mg mL−1 geneticin.

Protocol tips
The pKS2-ST::CBHII vector was used to transform competent cells of W. anomalus 54-A by electroporation, using the Gene Pulser electroporation system Xcell™ (Bio-Rad Laboratories) at 1400 V and 200 Ω. The W. anomalus 54-A clones were selected in YPD medium supplemented with 500 mg mL−1 geneticin.

Publication protocol

The pKS2-ST::CBHII vector was used to transform competent cells of W. anomalus 54-A by electroporation, using the Gene Pulser electroporation system Xcell™ (Bio-Rad Laboratories) at 1400 V and 200 Ω. The W. anomalus 54-A clones were selected in YPD medium supplemented with 500 mg mL−1 geneticin. Clones were confirmed by PCR using the primers 5′-GAGGAGAGCATAGAAATGGGG-3′ and 5′-CAGCAGTAGCCATAGCACCA-3′, which amplify a fragment from cbhII gene. The PCR-positive clones were evaluated at 55 mL scale, according to vector pKS2-ST manufacturer's instructions (Dualsystems Biotech). Briefly, each clone was incubated for 10 h in 10 mL YPD, after which 15 mL of fresh YPD medium was added. After 14 h incubation, 30 mL of fresh YPD medium was added to reach a final culture volume of 55 mL. After 6 h of incubation, it was expected that the glucose was exhausted, and this time was considered as the beginning of the induction phase. 1 mL aliquots were taken every 24 h for 96 h to measure extracellular enzyme activity and cell density. All the assays were performed in triplicate at 28°C and 180 rpm. Residual glucose quantitation was carried out by DNS method [17]. Crude extracellular fractions of the clone with the highest enzyme activity were loaded and processed by SDS-PAGE.

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