Team:NCTU Formosa/VP design

Violacein pathway

Design

We use the pathway which is mentioned by iGem 2010 team Slovenia, called the violacein biosynthesis pathway.
This is a spontaneous cascade pathway. 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. Later, VioE transforms the dimer to protodeoxyviolaceinic acid (PVA). The resulting PVA could be further converted into different products, such as deoxyviolacein catalyzed by VioC or protoviolaceinic acid catalyzed by VioD. The resulting protoviolaceinic acid can be converted into violacein by VioC again(Figure 1).

Circuit A

In circuit A, we used the constitutively expressing promoter, Ptet (BBa_R0040) to initiate the expression of vioA, vioB ,and vioE , which convert L-tryptophane into PVA.

Circuit B

In circuit B (BBa_K539461), the expression is promoted by Plac promoter (BBa_R0010) but the expression is restricted by 37oC RBS (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 (BBa_K539413) is translated. The VioD catalyze PVA into protoviolaceinic acid. The other translated sequence is tetR ( BBa_C0040 ), which repress the expression of circuit A that include Ptet (BBa_R0040). This way, Ecoli will focus on producing vioD instead of wasting resources on non-intended products.

Circuit C

The circuit C is regulated by heat sensitive cI QPI with high promoter (BBa_K098995). That is why vioC (BBa_K539513) is translated when over 42 oC condition, meanwhile LacI (BBa_C0012) and TetR (BBa_C0040) will also repress the expression of the other 2 circuits above. This way, Ecoli will focus on producing vioC instead of wasting resources on non-intended products, and we can even control the pathway as we wish.

Next, we will describe how our mechanism works: First, in order to accumulate enough precursor of PVA , we set the temperature at 30℃(Figure 5).

Second, we direct the pathway towards the production of Deoxychromoviridans, by simply rising the temperature to 42℃. At this moment, the constitutively produced CI inhibitor (BBa_K098995) will be degraded, therefore circuit C will be initiated and produce vioC, which catalyze PVA into deoxyviolacein that show dark purple pigment. At the same time, LacI (BBa_C0012)and tetR (BBa_C0040) will also show up and inhibit the production of circuit A and circuit B as well(Figure 6).

We also have the second option which directs the pathway towards the production of Protoviolaceinic acid. This can be done by simply rise the temperature to 37℃. At this moment, circuit B is translated because of the RNA thermometer (BBa_K115002) and produce vioD and tetR (BBa_C0040). Therefore vioD will then catalyze PVA into Protoviolaceinic acid, and at the same time tetR will inhibit the expression of circuit A. Moreover, the constitutively produced CI inhibitor (BBa_K098995) would repress the production of vioC(Figure 7). At this stage, our Ecoli will produce dark green color. Moreover, we can continue the pathway to obtain another product.

We can continue this pathway by rising the temperature even higher till it reach 42℃. At this stage, circuit C will be initiated. This circuit is promoted by CI repressed promoter, which will promote the expression only when the temperature reach 42℃ or higher (BBa_K098995) ,and the following vioc is then expressed. At this point, vioC will catalyze the accumulated Protoviolaceinic acid into Violacein(Figure 8). Simultaneously, our Ecoli will produce the enzyme to switch the color into dark purple.