Difference between revisions of "Part:BBa K4081994"
 
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<partinfo>BBa_K4081994 short</partinfo>
 
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GAPDHp
 
 
Generally, GAP promoter can drive the expression of glyceraldehyde-3-phosphate dehydrogenase gene in many species.
 
Generally, GAP promoter can drive the expression of glyceraldehyde-3-phosphate dehydrogenase gene in many species.
  
  
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<b><font size"3">Biology and Usage</font></b>
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===Usage and Biology===
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A lot of attention has been drawn to Glyceraldehyde-3-Phosphate Dehydrogenase (GAP) promoter. GAP mRNA accounts for 2–5% of the poly(A) RNA in yeasts, suggesting that it can be effectively used in the high-efficiency expression of transgenes[1]. In our project, we use GAP promoter to promote the expression of FKP. General vectors have been designed to rely on homologous GAPDH promoter regions to drive the expressions of heterologous genes in yeasts and other filamentous fungi.
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A lot of attention has been drawn to Glyceraldehyde-3-Phosphate Dehydrogenase (GAP) promoter. GAP mRNA accounts for 2–5% of the poly(A) RNA in yeasts, suggesting that it can be effectively used in the high-efficiency expression of transgenes[1]. In our project, we use GAP promoter to promote the expression of FKP in Saccharomyces cerevisiae BY4741. General vectors have been designed to rely on homologous GAP promoter regions to drive the expressions of heterologous genes in yeasts and other filamentous fungi.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4081994 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4081994 SequenceAndFeatures</partinfo>
  
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<b><font size"3">Design and Properties</font></b>
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We use GAP promoter to promote the expression of FKP.
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We get GAP promoter from vector PML104.
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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 GAP promoter: we transform the gene "GAP promoter and FKP ([https://parts.igem.org/Part:BBa_K4081992# BBa_K4081992])" 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 FKP by GAP 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">Reference</font></b>
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[1]Jia Y, Li S, Allen G, Feng S, Xue L. A novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter for expressing transgenes in the halotolerant alga Dunaliella salina. Curr Microbiol. 2012 May;64(5):506-13.
  
 
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Latest revision as of 08:01, 18 October 2021


GAP promoter

Generally, GAP promoter can drive the expression of glyceraldehyde-3-phosphate dehydrogenase gene in many species.


Biology and Usage

A lot of attention has been drawn to Glyceraldehyde-3-Phosphate Dehydrogenase (GAP) promoter. GAP mRNA accounts for 2–5% of the poly(A) RNA in yeasts, suggesting that it can be effectively used in the high-efficiency expression of transgenes[1]. In our project, we use GAP promoter to promote the expression of FKP in Saccharomyces cerevisiae BY4741. General vectors have been designed to rely on homologous GAP promoter regions to drive the expressions of heterologous genes in yeasts and other filamentous fungi.


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 GAP promoter to promote the expression of FKP.

We get GAP promoter from vector PML104.

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 GAP promoter: we transform the gene "GAP promoter and FKP (BBa_K4081992)" 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 FKP by GAP 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.


Reference

[1]Jia Y, Li S, Allen G, Feng S, Xue L. A novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter for expressing transgenes in the halotolerant alga Dunaliella salina. Curr Microbiol. 2012 May;64(5):506-13.