nguyenphihiep
Junior Member
DNA constructs.
Standard procedures for the isolation and manipulation of DNA were used as described in Ausubel et al. (1). The pDLG42-CSL1 plasmid (29) served as a template to amplify the E. coli tryptophanase (tnaA) gene using Phusion High-Fidelity DNA Polymerase (Finnzymes). For E. coli expression of proteins with a C-terminal six-histidine tag, we first cloned the tnaA gene into the pET-24(+) vector (Merck Biosciences) with the primers pET24-TnA-FWD (5′-TCTAGGATCCAAAATAAGGAGGAAAAACATATGAAGGATTATGTAATGGAAAACTTTAAAC-3′) and pET24-TnA-REV (5′-GTGCTCGAGAACTTCTTTCAGTTTTGCGGTGAAG-3′) using BamHI and XhoI restriction sites (underlined in both primers). The subsequent construct, designated pET-T, had an E. coli Shine Dalgarno sequence (in boldface) (24) to ensure efficient translation and an NdeI restriction site. The S. cerevisiae STR3 gene was amplified from genomic DNA from the wine yeast strain EC-1118 with primers pET24-STR3-FWD (5′-TCTACATATGCCGATCAAGAGATTAGATACA-3′) and pET24-STR3-REV (5′-GTGCTCGAGCAATTTCGAACTCTTAATATTCAATTCTGA-3′). The STR3 coding region (1,398 bp) was cloned using the NdeI and XhoI restriction sites (underlined in both primers) in the pET-T plasmid, thus yielding the pET-STR3 construct.
The pDLG42-PGK1 plasmid was constructed by cloning the 1.8-kb HindIII fragment released from the pHVXII plasmid containing the phosphoglycerate kinase I gene (PGK1) constitutive promoter (PGK1P) and terminator (PGK1T) cassettes, into the HindIII site of the yeast single-copy integrating plasmid pDLG42. This plasmid contains the ILV2 (SMR1-410) marker gene, which confers resistance to SMM. To clone STR3 into the pDLG42-pGK1 plasmid, the gene was amplified by PCR using pET-STR3 as a template and primers STR3-XhoI-FWD (5′-GACTCTCGAGATGCCGATCAAGAGATTAGATAC-3′), and STR3-XhoI-REV (5′-TAGCCTCGAGTTACAATTTCGAACTCTTAATATTC-3′), which were engineered to introduce an XhoI restriction site (underlined in both primers). The PCR product was cloned into the XhoI site of the pDLG42-PGK1 plasmid between the PGK1P and PGK1T sequences. The resulting plasmid, pDLG42-PGK1-STR3, was linearized with ApaI and transformed into VIN 13. Transformants were selected in SCD-SMM medium, genomic DNA was isolated, and the integration of the STR3 expression cassette into the genome was confirmed by PCR. This transformant was designated VIN 13 (STR3). The integrity of all expression constructs used in this study was confirmed by DNA sequencing using the Australian Genome Research Facility, Brisbane, Australia.
Standard procedures for the isolation and manipulation of DNA were used as described in Ausubel et al. (1). The pDLG42-CSL1 plasmid (29) served as a template to amplify the E. coli tryptophanase (tnaA) gene using Phusion High-Fidelity DNA Polymerase (Finnzymes). For E. coli expression of proteins with a C-terminal six-histidine tag, we first cloned the tnaA gene into the pET-24(+) vector (Merck Biosciences) with the primers pET24-TnA-FWD (5′-TCTAGGATCCAAAATAAGGAGGAAAAACATATGAAGGATTATGTAATGGAAAACTTTAAAC-3′) and pET24-TnA-REV (5′-GTGCTCGAGAACTTCTTTCAGTTTTGCGGTGAAG-3′) using BamHI and XhoI restriction sites (underlined in both primers). The subsequent construct, designated pET-T, had an E. coli Shine Dalgarno sequence (in boldface) (24) to ensure efficient translation and an NdeI restriction site. The S. cerevisiae STR3 gene was amplified from genomic DNA from the wine yeast strain EC-1118 with primers pET24-STR3-FWD (5′-TCTACATATGCCGATCAAGAGATTAGATACA-3′) and pET24-STR3-REV (5′-GTGCTCGAGCAATTTCGAACTCTTAATATTCAATTCTGA-3′). The STR3 coding region (1,398 bp) was cloned using the NdeI and XhoI restriction sites (underlined in both primers) in the pET-T plasmid, thus yielding the pET-STR3 construct.
The pDLG42-PGK1 plasmid was constructed by cloning the 1.8-kb HindIII fragment released from the pHVXII plasmid containing the phosphoglycerate kinase I gene (PGK1) constitutive promoter (PGK1P) and terminator (PGK1T) cassettes, into the HindIII site of the yeast single-copy integrating plasmid pDLG42. This plasmid contains the ILV2 (SMR1-410) marker gene, which confers resistance to SMM. To clone STR3 into the pDLG42-pGK1 plasmid, the gene was amplified by PCR using pET-STR3 as a template and primers STR3-XhoI-FWD (5′-GACTCTCGAGATGCCGATCAAGAGATTAGATAC-3′), and STR3-XhoI-REV (5′-TAGCCTCGAGTTACAATTTCGAACTCTTAATATTC-3′), which were engineered to introduce an XhoI restriction site (underlined in both primers). The PCR product was cloned into the XhoI site of the pDLG42-PGK1 plasmid between the PGK1P and PGK1T sequences. The resulting plasmid, pDLG42-PGK1-STR3, was linearized with ApaI and transformed into VIN 13. Transformants were selected in SCD-SMM medium, genomic DNA was isolated, and the integration of the STR3 expression cassette into the genome was confirmed by PCR. This transformant was designated VIN 13 (STR3). The integrity of all expression constructs used in this study was confirmed by DNA sequencing using the Australian Genome Research Facility, Brisbane, Australia.
