Team:NCTU Formosa/VP data

Violacein pathway

Data

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.13
   
   
   
   Figure 13. The vioD is cloned from vioABDE which is designed by 09 iGEM Cambridge.
   
   
2. vioC(BBa_K539513 )
   Figure 14.
   
   
   
   Figure 14. The vioC is cloned from vioABCDE which is designed by 09 iGEM Cambridge.
   
   
3. promoter (lacI regulated)+RNA thermometer(Part:BBa_K539421) Figure 15.
   
   
   
   Figure 15. Built in Plac and 37℃celcius regulator RBS
   
   
4. promoter (lacI regulated)+RNA thermometer +vioD(BBa_K539431)
   Figure 16.
   
   
   
   Figure 16. Built in Plac, 37℃celcius regulator RBS and vioD.
   
   
5. heat sensitive cI QPI with high promoter and a RBS(BBa_K539521)
   Figure 17.
   
   
   
   Figure 17. Built in a 42℃ device with a strong expressing RBS.
   
   
6. a RBS with vioC( BBa_K539522)
   Figure 18.
   
   
   
   Figure 18. Built in strong expressing RBS with vioC.
   
   
7. a RBS+ lacI repressor+ RNA thermometer+ tetR+ double terminator(BBa_K539558)
   Figure 19.
   
   
   
   Figure 19. Built in strong expressing RBS, LacI, 37’celcius regulator RBS, tetR and Terminator.
   
   
8. vioC+a RBS+lacI repressor+RNA thermometer+tetR+double terminator ( BBa_K539563)
   Figure 20.
   
   
   
   Figure 20. Built in vioC , strong expressing RBS, LacI, 37’celcius regulator RBS, tetR and terminator.
   

   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 interest part.
   The following figure shows the unsual gel electrophoresis result which the Xba I restriction site couldn't recognize the restriction site.
   

   
   
   Figure 21. Compare our part before and after digestion to show the point mutation of Xba I.
   

Discussion

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.

Firgure21. The overall conception of isobutanol biosynthesis pathway.

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) show 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 Ecoli 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 circuit we design explicate our concept of pathway commander, of which we can stop and direct the mechanism as we wish.