Team:NCTU Formosa/VP design

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Violacein pathway

Introduction

We use some parts which are from iGem 2009 Cambridge and iGEM 2010 Slovenia, called the Vio operon.
This is a spontaneous cascade pathway. The initial compound, L-tryptophane is catalyzed by VioA to indole-3-pyruvic acid imine (IPA imine) which then is converted into dimer by VioB. Later, VioE transforms the dimer to protodeoxyviolaceinic acid (PVA). The product, PVA, could be further converted into different compounds, such as deoxyviolacein catalyzed by VioC or protoviolaceinic acid catalyzed by VioD. The product, protoviolaceinic acid, can be converted into violacein by VioC (Figure 1). The branched pathway is made by enzymes’ reactions in different order. For instance, L-tryptophane has to be converted into deoxyviolacein by VioABEC, and into Violacein by VioABEDC. (Figure 1).

Figure 1. Violacein pathway

Design

We design three circuits (Figure 2, 3, 4) to obtain the chain mechanism. With the help of different RNA thermometers, we can decide the final product by placing the E.coli in different temperatures. And we can also stop the pathway in intermediate states.
Following is the three circuits:

Circuit A

Figure 2. Circuit A: Expression of vioA, vioB ,and vioE

In circuit A, we used the constitutive promoter, Ptet (BBa_R0010) to initiate the expression of vioA, vioB ,and vioE , which convert L-tryptophane into PVA.

Circuit B

Figure 3. Circuit B(BBa_K539461PSB1C3 ): Expression of vioD

In circuit B (BBa_K539461), is initiated by Plac promoter (BBa_R0010) but the expression is restricted by 37℃ RBS (BBa_K115002) , which means the ribosome will bind to the ribosome binding site only if the temperature reaches 37 ℃ or higher, and the vioD (BBa_K539413) is translated. The VioD catalyzes PVA into protoviolaceinic acid. The other gene tetR (BBa_C0012), which represses the expression of circuit A that includes Ptet (BBa_R0040). This way, E.coli will focus on producing VioD instead of wasting resources on non-intended products.

Circuit C

Figure 4. Circuit C: Expression of vioC.

The circuit C is regulated by heat-sensitive cI Quad-Part Inverter (QPI) with strong promoter (BBa_K098995). That is why vioC (BBa_K539513) is translated when over 42 ℃ condition, meanwhile LacI (BBa_C0012) and TetR (BBa_C0040) will also repress the expression of the other 2 circuits above. This way, E.coli 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).

Figure 5. By translating vioA, vioB and vioE at 30℃ we could obtain PVA as the end product.

Second, we direct the pathway towards the production of Deoxychromoviridans, by simply raising 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 catalyzes PVA into deoxyviolacein that shows dark purple. 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).

Figure.6 The VioC could catalyze PVA into Deoxychromoviridans at 42℃,and meanwhile the tetR would repress the Ptet contained circuit as well as the LacI repress the Plac.

We also have the second option which directs the pathway towards the production of Protoviolaceinic acid. This goal can be achieved by simply raise the temperature to 37℃. At this moment, circuit B is translated because of the RNA thermometer (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 (BBa_K098995) would repress the production of VioC (Figure 7). At this stage, our E.coli will produce dark green pigment. Moreover, we can continue the pathway to obtain another product.

Figure.7 The VioD could catalyze PVA into Protoviolaceinic acid at 37℃ ,at the same time the Ptet contained circuit would be repressed and the PCI contained circuit as well.

We can continue this pathway by raising the temperature even higher till it reaches 42℃. At this stage, circuit C will be initiated. This circuit will be turned on by CI repressed promoter, which will start the expression only when the temperature reaches 42℃ or higher (BBa_K098995) ,and the following vioD is expressed. At this point, VioC will catalyze the accumulated Protoviolaceinic acid into Violacein(Figure 8). Simultaneously, our E.coli will produce the enzyme to switch the color into dark purple.

Figure.8 The VioC could catalyze the Protoviolaceinic acid into the Violacein at 42℃ after accumulating it at 37℃, meanwhile, the tetR would repress the Ptet contained circuit as well as the LacI repress the Plac.