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Fig. 1 | Biotechnology for Biofuels

Fig. 1

From: A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

Fig. 1

Schematic representation of the reverse engineering approach for the introduction of the mutated alleles. a b show the direct replacement approach: a two cassettes were amplified, one containing the mutated allele and its terminator region amplified from genomic DNA of the corresponding mutant, flanked downstream by the restriction site for the I-SceI endonuclease and a synthetic homologous recombination sequence (SHR) [79]. A second cassette was amplified from plasmid pUG6 [80] for kanMX and from plasmid pUG-natNT2 for natNT2 containing the marker gene flanked upstream by the same SHR sequence as the first cassette and downstream by the restriction site for the I-SceI endonuclease and by 50 bp homologous to the region immediately downstream of the ORF. Upon co-transforming the two cassettes into S. cerevisiae, recombination at the SHR sequence results in integration in the genome and replacement of the wild-type allele. Transformants were selected in YPD containing G418 and/or nourseothricin. b Strains containing the marker gene were transformed with plasmid pUDE206 [81] expressing the I-SceI endonuclease and selected on YPD agar plates containing hygromycin. I-SceI cuts upstream and downstream of the marker gene, resulting in homologous recombination of the repeated terminator region, and thereby removal of the marker. Removal of marker genes was confirmed by the absence of growth on YPD agar plates containing G418 and/or nourseothricin. For removal of pUDE206, strains containing pUDE206 were grown on YPD and colonies were isolated on YPD agar plates. Plasmid removal was confirmed by the absence of growth on YPD agar plates containing hygromycin. c, d show the deletion/counter-selection approach: c two cassettes were amplified, one containing the GALp-GIN11M86 gene amplified from pGG119 [49], flanked upstream by 50 bp homologous to the ORF of the gene to be deleted. A second cassette was amplified from plasmid pUG-hphNT1 [82] containing the hphNT1 marker, flanked upstream by 50 bp homologous to the GALp-GIN11M86 gene and downstream by 50 bp homologous to the ORF to be deleted. Upon co-transforming the two cassettes into S. cerevisiae, they recombine at the SHR sequence insert into the genome, replacing the wild-type allele. d The mutated ORF of the gene was amplified from genomic DNA of the corresponding mutant and was used to transform the strain containing GALp-GIN11M86. Transformants were selected on YPGal agar plates. Replacement of the deleted allele was tested by the ability of transformants to grow in YPGal, inability to grow in the presence of hygromycin, as well as by PCR

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