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Strains used in this study.
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Strains used in this study.

Journal: Bioengineered

Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose

doi: 10.1080/21655979.2016.1207019

Figure Lengend Snippet: Strains used in this study.

Article Snippet: S. cerevisiae (BCRC 21685) was purchased from Bioresource Collection and Research Center, Food Industry Research and Development Institution, Hsinchu, Taiwan.

Techniques: Over Expression, Plasmid Preparation

Construction of deletion cassette with long flanking homology regions by overlapping PCR. In the first round of PCR, the upstream sequence of the nth1 gene, the kanr marker gene and the downstream sequence of the nth1 gene were amplified in 3 different PCR reaction tubes. S. cerevisiae genomic DNA was used as template for amplification of 0.26 kb 5′ UP nth1 region and 0.4 kb 3′ DOWN nth1 region by using primers L1, L2, and L3, L4, respectively. The pFA6a-kanMX6 vector was used as a template for amplification of kanMX region by using primers kanMX-F and kanMX-R. In the second round of PCR, the 5′ UP nth1 region and kanMX region were fused together by using primers L1 and kanMX-R. In the third round of PCR, the 3′ DOWN nth1 region and second round PCR product (5′ UP nth1 region-kanMX region) were fused together by using primers L1 and L4. The third round PCR product (5′ UP nth1 region- kanMX region - 3′ DOWN nth1 region) was cloned into pGEMT vector for construction of the disruption cassette. After transformation of the disruption cassette into S. cerevisiae, homologous recombination takes place with the deletion of target gene.

Journal: Bioengineered

Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose

doi: 10.1080/21655979.2016.1207019

Figure Lengend Snippet: Construction of deletion cassette with long flanking homology regions by overlapping PCR. In the first round of PCR, the upstream sequence of the nth1 gene, the kanr marker gene and the downstream sequence of the nth1 gene were amplified in 3 different PCR reaction tubes. S. cerevisiae genomic DNA was used as template for amplification of 0.26 kb 5′ UP nth1 region and 0.4 kb 3′ DOWN nth1 region by using primers L1, L2, and L3, L4, respectively. The pFA6a-kanMX6 vector was used as a template for amplification of kanMX region by using primers kanMX-F and kanMX-R. In the second round of PCR, the 5′ UP nth1 region and kanMX region were fused together by using primers L1 and kanMX-R. In the third round of PCR, the 3′ DOWN nth1 region and second round PCR product (5′ UP nth1 region-kanMX region) were fused together by using primers L1 and L4. The third round PCR product (5′ UP nth1 region- kanMX region - 3′ DOWN nth1 region) was cloned into pGEMT vector for construction of the disruption cassette. After transformation of the disruption cassette into S. cerevisiae, homologous recombination takes place with the deletion of target gene.

Article Snippet: S. cerevisiae (BCRC 21685) was purchased from Bioresource Collection and Research Center, Food Industry Research and Development Institution, Hsinchu, Taiwan.

Techniques: Sequencing, Marker, Amplification, Plasmid Preparation, Clone Assay, Transformation Assay, Homologous Recombination