Part:BBa_K4035011

Expression of the CUP1-(AP)7-CUP1 dimer on the extracellular membrane of S. cerevisiae

This part is the expression system of the CUP1 dimer (BBa_K4035004). 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-(AP)7-CUP1 (BBa_K4035004) 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_K4035004) 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 900
  Illegal PstI site found at 1122
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 900
  Illegal PstI site found at 1122
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 900
  Illegal PstI site found at 1122
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 900
  Illegal PstI site found at 1122
  Illegal AgeI site found at 150
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1116
  

Characterization

The characterization of this part can be found on the Part:BBa K4035004 page describing the dimer sequence.