Team:GDSYZX/Collection

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Collection

Among the parts we have contributed, UGT33 is the one we want to list into our “Collection”.
UGT33 is the UDP-glucose-4-O-glucosyltransferase(T8GT) protein coding region.UGT33 is the most active gene among R. rosea Tyrosol-Modifying UGTs. The protein it encodes reduces tyrosol to produce salidroside. UGT33 is used as the last gene to complete salidroside biosynthesis from tyrosine.

1. Vector Construction

As figure1, the carrier PUC-FLAG was digested and glued. From the results of running gel, the enzyme digestion products of the vector was 4000bp. As the result, we judge that we have target fragments.

figure 1

After the gene was transferred to the vector, we transferred the plasmid to e. coli overnight. The next day, PCR was performed to test whether the gene transfer was successful. The figure2 shows that the size of UGT33 gene is around 500dp, and the result is positive. However, the transfer of UGT33 into carrier was not smooth during the experiment. We started with the traditional enzyme training but it didn't work. Then we switched to a ligase, and it still was a false positive. Finally, we used the recombination link approach and successfully transferred UGT33 into the plasmid.

figure 2(PCR of the UGT33 bacterium solution）

2. Expression vs Time

In order to find a suitable detection time for salidroside, we explored the rule that the expression of 4HPAAS and UGT33 in Arabidopsis protoplasts in respond to the change of time. It was found that the expression of 4HPAAS and UGT33 reached the highest level 10 hours after transfection, so we speculated that this time should be the time with the highest synthesis rate of salidroside, so we chose 12 hours after transfection as the best time to detect salidroside.

A: Western blot was used to detect the expression of 4HPAAS and UGT33 in respond to the change of time;
B: We used “i mage J” to analyze the relative expression of 4HPAAS in respond to the change time (set the band gray value of 4hours as 1);
C: i mage J software and UGT33s with time (set the band gray value of 4 hours as 1).

3. Test expression products

As figure 3 shown, the protoplast and salidroside standard samples of Arabidopsis thaliana transfected with UGT33 and 4HPAAS genes were compared by liquid chromatography. The peak value of the product measured in sample No. 1 was basically consistent with the peak value of salidroside standard samples. Therefore, we can determine that the transfected protoplast of Arabidopsis thaliana can produce salidroside.

figure 3

4. Conclusion

Based on the above results, UGT33（and 4HPAAS） are the key genes for producing salidroside. In the case that the salidroside anabolism pathway in Arabidopsis has not been explored, we successfully predicted two key genes and obtained the expression of salidroside after transferring into Arabidopsis.

Part UGT33 modeling

I don’t know what to write here because EVERYTHING you want to see is in:
https://2019.igem.org/Team:GDSYZX/Model