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Fig. 1 | Biotechnology for Biofuels

Fig. 1

From: Fusion transcription factors for strong, constitutive expression of cellulases and xylanases in Trichoderma reesei

Fig. 1

Construction of the Xyr1-deficient T. reesei strain Xyr1′(81). a The uridine auxotrophic strain Δpyr4 was transformed with the plasmid pCD-Xyr1′(81)-HR, resulting in the insertion of a non-sense mutation (red line) and an adjacent BamHI restriction site in the xyr1 gene (blue arrow). The indicated flanking regions (green boxes) and the hygromycin resistance cassette (yellow arrow) were used for the homologous replacement strategy. Genomic coordinates are given on top. Position and orientation of the primers used for genomic testing are indicated by the short, black arrows. 5xf2, 5Xyr1_fwd2; Test_wt, Xyr1wt_Test_250rev; Test*, Xyr1*_Test_250rev. The thick, black line indicates the hybridization region for the probe used in the Southern blot assay. b Agarose gel electrophoresis of PCRs using the indicated primers and genomic DNA of indicated strains were performed to verify the complete replacement of the endogenous xyr1 gene. c A Southern blot analysis using BamHI-digested chromosomal DNA of the indicated strains and the indicated probe returned the expected signals at 6370 bp for Δpyr4 and 4170 bp and 2200 bp for Xyr′(81), along with an additional band above 10,000 bp indicating an ectopic insertion of the replacement cassette in Xyr1′(81) somewhere else in the genome

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