Difference between revisions of "Part:BBa K4035000"
 
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<partinfo>BBa_K4035000 short</partinfo>
 
<partinfo>BBa_K4035000 short</partinfo>
  
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This part is made of the Gal1 promoter (BBa_J63006), the A-agglutinin-binding subunit Aga2 (BBa_K416000) linked to the CUP1 copper metallotionein protein 1 (BBa_M45090) and the V5 tag (BBa_T2020).
 
  
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===USage and Biology===
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This part is the expression system of the CUP1 fused to Aga2 and tagged with a V5 epitope ([https://parts.igem.org/Part:BBa_K4035001 BBa_K4035001]). It has been made by inserting the endegenous CUP1 gene into the pCTcon2-V5 plasmid(1) and is composed of the Gal1 promoter ([https://parts.igem.org/Part:BBa_J63006 BBa_J63006]), the A-agglutinin-binding subunit Aga2 ([https://parts.igem.org/Part:BBa_K416000 BBa_K416000]) linked to the CUP1 copper metallothionein protein 1 ([https://parts.igem.org/Part:BBa_M45090 BBa_M45090]) and the V5 tag ([https://parts.igem.org/Part:BBa_T2020 BBa_T2020]).
  
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Copper metallotionein CUP1 (BBa_M45090) is a protein responsible for copper binding protein in the yeast Saccharomyces cerevisiae. Normally expressed intracellularly, CUP1 was fused to the A-agglutinin-binding subunit Aga2 (BBa_K416000) in order to be displayed on the outter yeast cell surface. This fusion protein also contained a V5 tag in order to check its expression by Western Blot and Immunostaining. The expression was under the control of the Gal1 promoter, so that the protein was expressed only in the presence of galactose.
 
  
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===Characterization===
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==Usage and Biology==
  
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===Sequence and Features===
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Copper metallothionein CUP1 ([https://parts.igem.org/Part:BBa_M45090 BBa_M45090]) is a protein responsible for copper binding protein in the yeast Saccharomyces cerevisiae. Normally expressed intracellularly, CUP1 was fused to the A-agglutinin-binding subunit Aga2 ([https://parts.igem.org/Part:BBa_K416000 BBa_K416000]) that attaches to the yeast cell wall through disulfide bonds to Aga1p. This leads to the presence of CUP1 on the outer membrane of the cell. The goal behind it was to improve the endogenous bio-accumulation property of wild type yeast by allowing it to bind more copper ions on its external surface.
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This fusion protein also contains a V5 tag ([https://parts.igem.org/Part:BBa_T2020 BBa_T2020]) in order to check its expression by Western Blot and Immunocytochemistry. The expression is under the control of the Gal1 promoter ([https://parts.igem.org/Part:BBa_J63006 BBa_J63006]), so that the protein is expressed only in the presence of galactose.
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==Sequence and Features==
 
<partinfo>BBa_K4035000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4035000 SequenceAndFeatures</partinfo>
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==Characterization==
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The characterization of this part can be found on the [https://parts.igem.org/Part:BBa_K4035001 Part:BBa K4035001] page that is describing the fusion protein Aga2-CUP1-V5.
  
  

Latest revision as of 20:28, 21 October 2021


Expression of CUP1 on the extracellular membrane of S. cerevisiae


This part is the expression system of the CUP1 fused to Aga2 and tagged with a V5 epitope (BBa_K4035001). It has been made by inserting the endegenous CUP1 gene into the pCTcon2-V5 plasmid(1) and is composed of the Gal1 promoter (BBa_J63006), the A-agglutinin-binding subunit Aga2 (BBa_K416000) linked to the CUP1 copper metallothionein protein 1 (BBa_M45090) and the V5 tag (BBa_T2020).


Usage and Biology

Copper metallothionein CUP1 (BBa_M45090) is a protein responsible for copper binding protein in the yeast Saccharomyces cerevisiae. Normally expressed intracellularly, CUP1 was fused to the A-agglutinin-binding subunit Aga2 (BBa_K416000) that attaches to the yeast cell wall through disulfide bonds to Aga1p. This leads to the presence of CUP1 on the outer membrane of the cell. The goal behind it was to improve the endogenous bio-accumulation property of wild type yeast by allowing it to bind more copper ions on its external surface.

This fusion protein also contains a V5 tag (BBa_T2020) in order to check its expression by Western Blot and Immunocytochemistry. The expression is under the control of the Gal1 promoter (BBa_J63006), so that the protein is expressed only in the presence of galactose.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

The characterization of this part can be found on the Part:BBa K4035001 page that is describing the fusion protein Aga2-CUP1-V5.