Fig. 3

Genomic insertion of Xyr1, XY1, and XZ0b expression cassettes. a The uridine auxotrophic strain Xyr1′(81) was transformed with the plasmids pRP4-TX(WT), pRP4-TXY(1), or pRP4-TXZ(0b) resulting in the targeted integration of the respective expression cassettes (blue arrow and blue, dashed lines) into the pyr4 locus using the pyr4 gene (orange arrow) and its promoter (orange line) as auxotrophic marker. The gray boxes represent the flanking regions used for the homologous recombination strategy. The wild-type pyr4 locus is depicted for comparison only. Position and orientation of the primers used for genomic testing are indicated by the short, black arrows. 5pf3, 5pyr4_fwd3; Ptr, Ptef_rev-BspTI; p3f, pyr4_3fwd; Tpr2, Tpyr4_rev2. The thick, black line indicates the hybridization region for the probe used in the Southern blot assay. Recognition sites for the restriction endonuclease SpeI are depicted. b Agarose gel electrophoresis of PCRs using the indicated primers and genomic DNA of indicated strains were performed to verify the integration of the TF expression and pyr4 re-establishment cassettes into the pyr4 locus. c A Southern blot analysis using SpeI-digested chromosomal DNA of the indicated strains and the indicated probe returned the expected signals at 2501 bp for Xyr1′(81) and 6670 bp, 6355 bp, and 6274 bp for TX(WT), TXY(1), and TXZ(0b), respectively