Resveratrol

Results

Biobrick

We succeded in the cloning and sequencing of our two biobricks, 4-Coumarate ligase from arabidophsis thaliana and Stilbene synthase from vitis vinifiera with the RBS B0034 that should work in various organisms, lactobacillus and e-coli. Sequencing was done at GATC biotech and Uppsala Genome center using sanger sequencing.

We also succeded in the making of our operon containing 4Cl and STS together.

BBa_K1033000 - tyrosine ammonia lyase (TAL) with RBS

BBa_K1033001 - 4 coumarate ligase (4CL) with RBS

BBa_K1033002 - stilbene synthase (STS) with RBS

BBa_K1033003 - B0034-4Cl-B0034-STS

Western blot

We also succeeded in expressing the enzyme stilbene synthase in E. coli. Although our expression of the protein was very weak, and due to time constraints we were not able to optimize our experiment.

To enable the detection of this protein by anti-his antibodies, 6-histidine tags was incorporated in the sequence. This way we could detect our enzyme with anti-his antibodies.

We expressed our protein with a multipromotor working in both Lactobacillus and E. coli. This way, we can easily transfer stilbene synthase to Lactobacillus later on.

The size of our protein was calculated using ProtParam ^[5], 43 kDA.

High pressure liquid chromatography

We tested our biobrick 4Cl-STS on HPLC, by adding p-coumaric acid as a precursor. The result we saw was quite unclear. We saw that the E. coli produced something out of the ordinary, but the absorbation was low and the peaks did not exactly match the standard. The peak at around ~33 min could correspond to our standard, but it is unclear. We theorize it is something in the actual hplc measurement that fails, or that something happens to our resveratrol metabolite in our E. coli.

4Cl-STS translational fusion expressed in e-coli

Figure 1: E-coli supposed to produce resveratrol. As we can see, we got very low concentration peaks at ~30 min, ~33 min and ~36 min.


Resveratrol standard

Figure 2: Resveratrol standard that is scaled down to correspond to the absorbations of our e-coli supposed to produce the corresponding metabolite. The peaks are at around ~33 and ~34.