Resveratrol

Results

Summary

Although we managed to clone out and sequence verify the genes for resveratrol production, we have had some problems in the characterization. The results are unclear, and we did not have time for further investigations.For detailed information about the characterisation methods, see the protocol section

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. However, 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, a 6-histidine tag was incorporated in the sequence. This way we could detect our enzyme with anti-his antibodies.

We expressed our protein with a promotor 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] to 43 kDA.


Figure 1: Number 2 shows a very weak band of our protein at around 43 kDA.
Positive control -> 1, Stilbene synthase -> 2

High pressure liquid chromatography

We tested our biobrick 4Cl-STS on HPLC where we tried to detect resveratrol production in the E. coli strains. By adding p-coumaric acid as a precursor, we predicted that resveratrol would be produced. The result we saw was quite unclear. We saw that the E. coli produced something out of the ordinary, but the absorbtion 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 that it is something in the actual HPLC measurement that fails, or that something disturbs our resveratrol metabolite in our E. coli. This result could correspond to the poor results in our westernblot. We hope that iGEM teams can continue to work on these biobricks in the future to undoubtedly characterize them as resveratrol producing.

4Cl-STS translational fusion expressed in e-coli
Figure 2: E. coli supposed to produce resveratrol. As we can see, we got very low absorbance peaks at ~30 min, ~33 min and ~36 min. Measurements before ~10 min (left side of graph) are excluded in the chromatogram do to noisy data in these low absorbance levels.


Resveratrol standard
Figure 3: Resveratrol standard, peaks with retention times around 33 and 34 min.


Resveratrol standard scaled
Figure 4: 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.


Lysed bacterial culture without plasmid of assembly

Figure 5: E. coli culture injected to the HPLC without our biobrick tyrosine ammonia lyase. Here we can see that there is originally no peaks around 30-35 minutes.

References