Fig. 3

Genomic insertion of the SynX expression cassette. a The uridine auxotrophic strain Xyr1′(81) was transformed with the plasmid pRP4-SynX resulting in the targeted integration of the SynX expression cassette (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 grey boxes represent the flanking regions used for the homologous recombination. The wild-type (WT) pyr4 locus is depicted for comparison and the chromosomal coordinates according to [74] are given. Position and orientation of the primers used for genotype testing are indicated by short, black arrows. 5pf3, 5pyr4_fwd3; Tpr2, Tpyr4_rev2; Ptr, Ptef_rev-BspTI; p3f, pyr4_3fwd. 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 the amplification products from PCR assays using the indicated primer pairs and DNA samples of indicated strains. c A Southern blot analysis using SpeI-digested chromosomal DNA of the indicated strains returned the expected signals at 2501 bp for Xyr1′(81) and 7324 bp for TXYE and verifies the exclusive integration of the expression cassette at the pyr4 locus. L, DNA size ladder