P_GAP

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	Each plasmid was amplified in E. coli, linearized with BglII or SmiI (Fermentas) and transformed into the corresponding P. pastoris strains. Single colonies were transferred to 96-well deep-well plates for standard cultivation as described previously [42]. To compare the expression levels, GFP fluorescence was measured as described by [47].

Protocol tips
Each plasmid was amplified in E. coli, linearized with BglII or SmiI (Fermentas) and transformed into the corresponding P. pastoris strains. Single colonies were transferred to 96-well deep-well plates for standard cultivation as described previously [42]. To compare the expression levels, GFP fluorescence was measured as described by [47].

Publication protocol

"Genomic DNA was isolated from each strain using DNAeasy kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol for large scale yeast DNA isolation. All targeted loci in the P. pastoris genome were PCR amplified and sequenced to confirm expected knock-outs using primers described in Table S1 and Phusion™ High-Fidelity DNA-polymerase according to manufacturer’s recommendations. Southern blotting and hybridization were carried out according to standard protocols [45] to verify the correct integration of the excision cassettes. PCR amplification of the probes specific to the Zeocin™ gene and knock-out regions in genes AOX1, HIS4 and KU70 was performed using DIG-labeled dNTPs (Roche, Basel, Switzerland) and AmpliTaqGOLD® (Roche, Basel, Switzerland) polymerase according to the manufacturers’ instructions. Primer details can be found in Table S1.

Correct plasmid sequences were confirmed by Sanger sequencing. For testing the functionality, an improved version of green fluorescent protein [46] was cloned into the multiple cloning site of each plasmid. Correct insertion was verified by sequencing. Each plasmid was amplified in E. coli, linearized with BglII or SmiI (Fermentas) and transformed into the corresponding P. pastoris strains. Single colonies were transferred to 96-well deep-well plates for standard cultivation as described previously [42]. To compare the expression levels, GFP fluorescence was measured as described by [47]."

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