pUC19-Pgpd-xyn2-Tgpd

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	Protoplast transformation of T. reesei was performed by using the polyethylene glycol method as described in Punt et al.[32].

Protocol tips
Protoplast transformation of T. reesei was performed by using the polyethylene glycol method as described in Punt et al.[32].

Publication protocol

Protoplast transformation of T. reesei was performed by using the polyethylene glycol method as described in Punt et al.[32]. Lysing enzymes from Trichoderma harzianum (Sigma-Aldrich) were used in the T. reesei protoplast preparation. For the transformation, the expression cassettes were released from the plasmids through digestion with appropriate restriction enzyme pairs, then purified, and mixed with equal amounts of plasmid pAN7-1. The mixture was then used for co-transformation of T. reesei protoplasts. Candidate transformants were streaked twice on PDA plates that contained 100 μg/ml of hygromycin B, and then transferred to PDA plates to form conidia. For each expression cassette, twenty single colonies were selected for cultivation in Mandels medium supplemented with 4% glucose, and the activity of xylanase in the supernatant was analyzed. For each expression cassette, the single colony that exhibited the highest productivity was selected for further study.

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