Part:BBa_K539461

promoter (lacI regulated)+RNA thermometer+vioD with RNA thermometer+tetR+double terminator

Please refer to the wiki for our overall concept:[http://2011.igem.org/Team:NCTU_Formosa/VP_design 2011 NCTU_Formosa]

We use some parts which are from iGem 2009 Cambridge, called the Vio operon.

This pathway is a spontaneous cascade pathway which the initial compound, L-tryptophane is catalyzed by Vio A to indole-3-pyruvic acid imine (IPA imine) which then is converted into dimer by Vio B.
VioE then transforms the dimer to protodeoxyviolaceinic acid (PVA).
The resulting PVA could then further be converted into different products, such as deoxyviolacein which is catalyzed by VioC and the other one is protoviolaceinic acid by VioD.
The resulting protoviolaceinic acid can then be converted into violacein by VioC.
We design our circuits this way to obtain the chain mechanism which at the end of the mechanism we have the accumulated protoviolaceinic acid. We then convert these PVA into violacein by using temperature controlled vioC expression.

The mechanism of Circuit B in our design:

Circuit B is translated because of the RNA thermometer (Part:BBa_K115002) and produces VioD and TetR (BBa_C0040). Therefore, VioD will then catalyze PVA into Protoviolaceinic acid, meanwhile, TetR will inhibit the expression of circuit A. Moreover, the constitutively produced CI inhibitor (Part:BBa_K098995) would repress the production of VioC. At this stage, our E.coli will produce dark green pigment. Moreover, we can continue the pathway to obtain another product.

Circuit B

Figure.1 Circuit B (Part:BBa_K539461 ,DH5α,PSB1C3): The expression of vioD

In circuit B (Part:BBa_K539461), the expression is initiated by Plac promoter (Part:BBa_R0010) but the expression is restricted by 37oC RBS (Part:BBa_K115002) which means, the ribosome will bind to the ribosome binding site only if the temperature reach 37 oC or higher, and the vioD (Part:BBa_K539413) is translated.
The VioD catalyzes PVA into protoviolaceinic acid. The other gene is tetR (Part:BBa_C0040), which represses the expression of circuit A that includes Ptet (Part:BBa_R0040). This way, E.coli will concentrate on producing VioD instead of wasting resources on unwanted product.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2119
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1194
  Illegal SapI.rc site found at 1269