Part:BBa_K323132

VioC, VioD and VioE enzymes fused with zinc fingers under pBAD promoter

The part contains three of the enzymes of the violacein biosynthetic pathway, fused to zinc finger proteins. VioC enzyme is linked to the Gli1 zinc finger (Part:BBa_K323086), vioD to HivC zinc finger (Part:BBa_K323087) and VioE enzyme to the PBSII zinc finger (Part:BBa_K323131). When translated to protein, three functional chimeric proteins which bind to a specific DNA sequence are produced. We designed a DNA program (Part:BBa_K323066) with these sequences to bind the chimeric enzymes to a closer proximity.

In combination with Part:BBa_K323135, we have bound all of the enzymes, necessary for synthesis of violacein, on the DNA program. This enabled a faster and increased synthesis of the desired product, as the enzymes were fixed close together and the intermediates were immediately available to the next enzyme.

We have designed another DNA program (Part:BBa_K323046) with no binding site for the HivC_VioD chimeric protein. By this we showed that the program determines the final product - in our case, larger amounts of deoxyviolacein were formed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1204
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3078
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 3177
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  Illegal SapI.rc site found at 4320
  Illegal SapI.rc site found at 4395