Difference between revisions of "Part:BBa K4081990"

 
 
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<partinfo>BBa_K4081990 short</partinfo>
 
<partinfo>BBa_K4081990 short</partinfo>
  
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ADH1p
 
  
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<b><font size"3">Biology and Usage</font></b>
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===Usage and Biology===
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ADH1 promoter is the yeast promoter for Alcohol Dehydrogenase I. It can be suppressed by ethanol. The full-length version is strong and promotes high expression. Truncated promoters are constitutive and have low expression. It can be effectively used in the high-efficiency expression of transgenes. In our promoter, we use ADH1 promoter to promote the expression of FucT2 in Saccharomyces cerevisiae BY4741.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4081990 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4081990 SequenceAndFeatures</partinfo>
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<b><font size"3">Design and Properties</font></b>
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We use ADH1 promoter to promote the expression of FucT2.
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We get ADH1 promoter from vector pAUR123.
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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 ADH1 promoter: we transform the gene "ADH1 promoter and FucT2 ([https://parts.igem.org/Part:BBa_K4081998# BBa_K4081998])" 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 FucT2 by ADH1 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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<b><font size"3">Experimental approach</font></b>
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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”.
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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.
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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.
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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.
  
  

Latest revision as of 08:02, 18 October 2021


ADH1 promoter


Biology and Usage

ADH1 promoter is the yeast promoter for Alcohol Dehydrogenase I. It can be suppressed by ethanol. The full-length version is strong and promotes high expression. Truncated promoters are constitutive and have low expression. It can be effectively used in the high-efficiency expression of transgenes. In our promoter, we use ADH1 promoter to promote the expression of FucT2 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 ADH1 promoter to promote the expression of FucT2.

We get ADH1 promoter from vector pAUR123.

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 ADH1 promoter: we transform the gene "ADH1 promoter and FucT2 (BBa_K4081998)" 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 FucT2 by ADH1 promoter(figure1).

Figure 1. 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.