Difference between revisions of "Part:BBa K4081995"
 
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We use TEF1 promoter to promote the expression of LAC12.
 
We use TEF1 promoter to promote the expression of LAC12.
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We get TEF1 promoter from the genome of Saccharomyces cerevisiae BY4741.
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[[Image:T--Jianhua--promoter.png|300px|thumb|center|Figure 1. Gel electrophoresis results of PCR promoter. Lane 1: Marker; Lane 2: GAP promoter(700bp); Lane 3: TEF1 promoter(577bp); Lane 4: ADH1 promoter(461bp).]]
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We tested the TEF1 promoter: we transform the gene "TEF1 promoter and LAC12 ([https://parts.igem.org/Part:BBa_K4081996# BBa_K4081996])" into Saccharomyces cerevisiae BY4741 by lithium acetate conversion method to integrate the genes into the genome of BY4741 for expression.The result of SDS-PAGE showed that we successfully expressed LAC12 by TEF1 promoter(figure1).
 
We tested the TEF1 promoter: we transform the gene "TEF1 promoter and LAC12 ([https://parts.igem.org/Part:BBa_K4081996# BBa_K4081996])" into Saccharomyces cerevisiae BY4741 by lithium acetate conversion method to integrate the genes into the genome of BY4741 for expression.The result of SDS-PAGE showed that we successfully expressed LAC12 by TEF1 promoter(figure1).
  
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[[Image:T--Jianhua--The result of SDS-PAGE.png|300px|thumb|center|Figure 1. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).]]
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[[Image:T--Jianhua--The result of SDS-PAGE.png|300px|thumb|center|Figure 2. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).]]
  
 
<b><font size"3">Experimental approach</font></b>
 
<b><font size"3">Experimental approach</font></b>
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4.Extract yeast total protein. Use SDS-PAGE to test whether the three proteins(FKP, LAC12, FucT2) are successfully expressed.
 
4.Extract yeast total protein. Use SDS-PAGE to test whether the three proteins(FKP, LAC12, FucT2) are successfully expressed.
  
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<b><font size"3">References</font></b>
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[1]Yu, S. ,  Liu, J. J. ,  Yun, E. J. ,  Kwak, S. ,  Kim, K. H. , &  Jin, Y. S. . (2018). Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered saccharomyces cerevisiae. Microbial Cell Factories,  17.
  
 
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Latest revision as of 07:59, 18 October 2021


TEF1 promoter


Biology and Usage

TEF1 promoter is the yeast transcription elongation factor promoter. It drives constitutive expression of genes, and is similar to mammalian EF1a promoter. It can be effectively used in the high-efficiency expression of transgenes. In our promoter, we use TEF1 promoter to promote the expression of LAC12 in Saccharomyces cerevisiae BY4741.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design and Properties

We use TEF1 promoter to promote the expression of LAC12.

We get TEF1 promoter from the genome of Saccharomyces cerevisiae BY4741.

Figure 1. Gel electrophoresis results of PCR promoter. Lane 1: Marker; Lane 2: GAP promoter(700bp); Lane 3: TEF1 promoter(577bp); Lane 4: ADH1 promoter(461bp).


We tested the TEF1 promoter: we transform the gene "TEF1 promoter and LAC12 (BBa_K4081996)" into Saccharomyces cerevisiae BY4741 by lithium acetate conversion method to integrate the genes into the genome of BY4741 for expression.The result of SDS-PAGE showed that we successfully expressed LAC12 by TEF1 promoter(figure1).

Figure 2. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).

Experimental approach

1.Construct recombinant plasmids. Get GAP promoter from vector PML104. Get TEF1 promoter from the genome of Saccharomyces cerevisiae BY4741. Get ADH1 promoter from vector pAUR123. Company synthetic genes of FKP, LAC12 and FucT2. Use vector pAUR123 to construct our plasimd “pAUR123-pGAP-FKP-pADH1-FucT2-pTEF1-LAC12”.

2.Transform the product (2.5μL) into DH5α competent cells (50μL), grow cells on agar plates (containing Ampicillin). Incubate plates at 37°C overnight. Colonies were screened by colony PCR and then grown at 37℃, 200rpm. Plasmids were extracted and sent for sequencing.

3.PCR the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene AurR from the plasmid with homology arms of BY4741. Transform it into BY4741 by lithium acetate conversion method to integrate genes into the genome of BY4741 for expression. Screen for transformants by AbA-YPD selection medium.

4.Extract yeast total protein. Use SDS-PAGE to test whether the three proteins(FKP, LAC12, FucT2) are successfully expressed.


References

[1]Yu, S. , Liu, J. J. , Yun, E. J. , Kwak, S. , Kim, K. H. , & Jin, Y. S. . (2018). Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered saccharomyces cerevisiae. Microbial Cell Factories, 17.