Part:BBa_K289003

GSH

According to the Toxicity mechanism, Adda binding site of MCs has a strong affinity to the lyophobic groove of PP1. Also, according to the Detoxicity mechanism, GSH could construct a covalent bond with Mdha banding site of MCs easily under the function of GST(Glutathione S-Transferase Agents which presents in the cell of Yeast) In our project, we connect PP1 gene after DNA-BD in the plasmid pGBKT7 and GSH gene after AD in the plasmid pGADT7, and then we transform these two plasmids into Yeast(AH109) so that it can express protein DNA-BD-PP1 and AD-GSH. At last our hero MCs enters. It will connect with AD-GSH which has a system can lead it to the nucleus of yeast, and then AD-GSH-MCs comes into the core to connect with DNA-BD-PP1 on chromosome. Expression of both fusion proteins in yeast and interaction between bait and prey indeed reconstituted a functional Gal4 transcription factor from the two separate polypeptides. Gal4 then recruited RNA polymerase II, leading to transcription of a GAL1-lacZ fusion gene. This reporter gene encodes the enzyme beta-galactosidase which labels the yeast cell when using a colorimetric substrate To active Gal4 promoter, two domains must come to each other close enough in the nucleus of Yeast. One is AD(Activation domain), the other is DNA-BD(DNA Binding domain). When AD and DNA-BD are close enough to each other, the Report genes transcription LacZ will be started. Then we use X-gal to detect whether LacZ is expressed( LacZ could turn the color of X-gal from white to blue).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 75
  Illegal EcoRI site found at 1097
  Illegal PstI site found at 99
  Illegal PstI site found at 1044
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 75
  Illegal EcoRI site found at 1097
  Illegal PstI site found at 99
  Illegal PstI site found at 1044
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 75
  Illegal EcoRI site found at 1097
  Illegal XhoI site found at 93
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 75
  Illegal EcoRI site found at 1097
  Illegal PstI site found at 99
  Illegal PstI site found at 1044
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 75
  Illegal EcoRI site found at 1097
  Illegal PstI site found at 99
  Illegal PstI site found at 1044
  
• 1000
  COMPATIBLE WITH RFC[1000]