Difference between revisions of "Part:BBa K4035009"

Revision as of 21:10, 20 October 2021

Expression of the CUP1-(GGGGS)3-CUP1 dimer on the extracellular membrane of S. cerevisiae

This part is made of the Gal1 promoter (BBa_J63006), the A-agglutinin-binding subunit Aga2 (BBa_K416000) linked to the dimer protein CUP1-(GGGGS)3-CUP1 (BBa_K4035002) 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_K4035002) 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
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1000
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 K4035002 page as this one is describing its expression system.

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 ==Usage and Biology==
-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 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.
+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_K4035002 BBa_K4035002]) 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.
 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.