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Table 4 Cloning strategy for the construction of yeast integrating plasmids YCt XRWt/Gm XDH/XKS1 and YCt XRDm/Gm XDH/XKS1. The shown primer sets were used to amplify the target sequences from the template plasmids. The amplification products were cloned into the corresponding restriction sites of the target plasmid.

From: Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae

  Target sequence Template plasmid Primers (restriction sites underlined) Target plasmid Restriction sites Resulting plasmid
1 Ct XRWt gene pET11-Ct XRWt Fwd:
5'-GGTGGTGGATCC ATGAG
CGCAAGTATCCGAGAC-3'
pRS416GPD BamH I
Sal I
pRS416GPD-Ct XRWt
  Ct XRDm gene pET11-Ct XRDm Rev:
5'CTAGTGGGTCGAC TTAAAC
GAAGATTGGAATGTTGTC-3'
pRS416GPD BamH I
Sal I
pRS416GPD-Ct XRDm
  Gm XDH gene pBTac1 Fwd:
5'-GGTGGTGGATCC ATGTCT
ACTCCTGAAAACTTATCT-3'
pRS416GPD BamH I
Sal I
pRS416GPD-Gm XDH
    Rev:
5'-CTAGTGGGTCGAC TTAC
TCAGGGCCGTTAATGATG-3'
   
  XKS1 gene Genomic S. cerevisiae DNA Fwd:
5'-GGTGGTGGATCC ATGTTG
TGTTCAGTAATTCAGAGA-3'
pRS416GPD BamH I
Sal I
pRS416GPD-XKS1
    Rev:
5'-GGTGGTGTCGAC TTAGAT
GAGAGTCTTTTCCAGTTC-3'
   
2 Gene cassettea XKS1 pRS416GPD-XKS1 Fwd:
5'-CATGGTGACGTC AGTTTATC
ATTATCAATACTCGCCATTTC-3'
YiP5 Aat II YXKS1
    Rev:
5'-GGTGGTGACGTC GGCCGCA
AATTAAAGCCTTCG-3'
   
3 Gene cassette Gm XDH pRS416GPD-Gm XDH Fwd:
5'-CATGGTATCGAT AGTTTATC
ATTATCAATACTCGCCATTTC-3'
YXKS1 Cla I YGm XDH/XKS1
    Rev:
5'-GGTGGTATCGAT GGCCGCA
AATTAAAGCCTTCG-3'
   
4 Gene cassette Ct XRWt pRS416GPD-Ct XRWt Fwd:
5'-GGTGGTGAATTC AGTTTATC
ATTATCAATACTCGCCATTTC-3'
YGm XDH/XKS1 EcoR I YCt XRWt/Gm XHD/XKS1
  Gene cassette Ct XRDm pRS416GPD-Ct XRDm Rev:
5'-GGTGGTGAATTC GGCCGCA
AATTAAAGCCTTCG-3'
YGm XDH/XKS1 EcoR I YCt XRDm/Gm XHD/XKS1
  1. a Gene cassettes contain S. cerevisiae TDH3 promoter (labeled GPD in the Table) – target gene – S. cerevisiae CYC1 terminator