Difference between revisions of "Part:BBa K4035013"
 
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<partinfo>BBa_K4035013 short</partinfo>
 
<partinfo>BBa_K4035013 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 dimer protein CUP1-(EAAAK)4-CUP1 (BBa_K4035006) 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 dimer ([https://parts.igem.org/Part:BBa_K4035006 BBa_K4035006]). It has been made by inserting the sequence 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 dimer protein CUP1-(EAAAK)4-CUP1 ([https://parts.igem.org/Part:BBa_K4035006 BBa_K4035006]) 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. In order to increase the copper retrieval efficiency, two copies of CUP1 were linked together and expressed at the outter surface of S. cerevisiae. Aga2 attaches to the yeast cell wall through disulfide bonds to Aga1p. The fusion of the dimer and Aga2 then leads to the presence of the CUP1 dimer on the outter membrane of the cell. 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. In order to increase the copper retrieval efficiency, two copies of CUP1 were linked together ([https://parts.igem.org/Part:BBa_K4035006 BBa_K4035006]) and expressed on the external surface of S. cerevisiae by fusion with the A-agglutinin-binding subunit Aga2 ([https://parts.igem.org/Part:BBa_K416000 BBa_K416000]). Aga2 attaches to the yeast cell wall through disulfide bonds to Aga1p. The fusion of the dimer and Aga2 then leads to the presence of the CUP1 dimer 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_K4035013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4035013 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_K4035006 Part:BBa K4035006] page describing the dimer sequence.
  
  

Latest revision as of 20:59, 21 October 2021


Expression of the CUP1-(EAAAK)4-CUP1 dimer on the extracellular membrane of S. cerevisiae


This part is the expression system of the CUP1 dimer (BBa_K4035006). It has been made by inserting the sequence 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 dimer protein CUP1-(EAAAK)4-CUP1 (BBa_K4035006) 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. In order to increase the copper retrieval efficiency, two copies of CUP1 were linked together (BBa_K4035006) and expressed on the external surface of S. cerevisiae by fusion with the A-agglutinin-binding subunit Aga2 (BBa_K416000). Aga2 attaches to the yeast cell wall through disulfide bonds to Aga1p. The fusion of the dimer and Aga2 then leads to the presence of the CUP1 dimer 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
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1218
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1218
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1218
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1218
    Illegal AgeI site found at 150
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

The characterization of this part can be found on the Part:BBa K4035006 page describing the dimer sequence.