Team:NCTU Formosa/VP data

Violacein pathway

Data

We also characterise the biobrick of BBa_K274004

Firstly ,we found out that there's a point mutation in this plasmid. This mutation occurs in the Xba I restriction site which takes a lot of time for us just to digest it in order to ligase this part with our interested parts.
The following figure shows the unusual gel electrophoresis result which the Xba I restriction enzyme couldn't recognize the restriction site. As it is shown in the figure above, it is very obvious that the band is on the same position as the undigested one.

In order to correct the mutation, we then design a primer as follows:

This primer include the precise sequence of the Xba I restriction site. We then do Polymerase Chain Reaction to extend the remaining sequence and amplify the exact plasmid that we want.

Last and the most important, we also notice something that this part is actually vioABDE instead of vioABCE. In previous time, we failed in every attempts to clone out vioC from this part. We then decided to do DNA sequencing to make sure every single sequence is correct. Afterward, we found the exact sequence of vioABDE instead of vioABCE.

So far, we have already partially finished assembling our circuits. To achieve our goal, we will try hard in remaining days.
The following is the part we finished:

1. vioD(BBa_K539413)
   
   
   
   Figure 16. The vioD is cloned from vioABDE which is designed by 09 iGEM Cambridge.
   
   
2. vioC(BBa_K539513 )
   
   
   
   Figure 17. The vioC is cloned from vioABCDE which is designed by 09 iGEM Cambridge.
   
   
3. promoter (lacI regulated)+RNA thermometer(BBa_K539421 )
   
   
   
   Figure 18. Built in Plac and 37℃celcius regulator RBS
   
   
4. promoter (lacI regulated)+RNA thermometer +vioD(BBa_K539431)
   
   
   
   Figure 19. Built in Plac, 37℃ regulator RBS and vioD.
   
   
5. heat sensitive cI QPI with high promoter and a RBS(BBa_K539521)
   
   
   
   Figure 20. Built in a 42℃ device with a strong expressing RBS.
   
   
6. a RBS with vioC( BBa_K539522)
   
   
   
   Figure 21. Built in strong expressing RBS with vioC.
   
   
7. a RBS+ lacI repressor+ RNA thermometer+ tetR+ double terminator(BBa_K539558)
   
   
   
   Figure 22. Built in strong expressing RBS, LacI, 37℃ regulator RBS, tetR and Terminator.
   
   
8. vioC+a RBS+lacI repressor+RNA thermometer+tetR+double terminator ( BBa_K539563)
   
   
   
   Figure 23. Built in vioC , strong expressing RBS, LacI, 37℃ regulator RBS, tetR and terminator.
   

Conclusion

According to the steps mentioned above, we could then control the branched pathway successfully by inserting those genes, which code for thermal regulation, into an E.coli. We then are able to construct a human regulated pathway which was considered to be spontaneous mechanism and to prove our concept is practicable.

The regulation is tested in violacein biosynthesis pathway, which allows us to thermally regulate the mechanism, and could also be monitored by color alteration resulting from the conversion.

Through color alteration, we could obviously observe different products as effect of different temperatures we set. When the temperature is maintained at 30℃, the colorless product called Protodeoxyviolaceinic acid(PVA) shows up, which means we stop the mechanism in this step and accumulate the product until it is enough to continue the branched mechanism. Then, If we raise the temperature up to 42℃ directly, the PVA would be catalyzed to Deoxychromoviridans by VioC. At the same time, our Ec.oli will turn into dark purple. On the other hand, if we raise the temperature up to 37℃ first, we would obtain dark green product called Protoviolaceinic acid (catalyzed by VioD). And if we put the E.coli in 42℃ after placing in 37℃ for a while , the resulting product we obtain is Violacein(catalyzed by VioC) which shows violet(Figure 9).

The circuits we design explicate our concept of pathway commander, of which we can stop and direct the mechanism as we wish.