Difference between revisions of "Part:BBa K4236101"

Revision as of 13:22, 12 October 2022

T7-RBS-His-atUGT+EGFP

The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS, and UGT. UGT is an enzyme catalyzing kaempferol into astragalin, and is the largest one among the three enzymes we used in our pathway (F3H, FLS, UGT). We firstly constructed an AtUGT + EGFP vector to test the expression of the UGT gene.

Usage and Biology

At this year's iGEM competition, we focused on Alzheimer's disease (AD), a neurodegenerative disease, and identified astragalin as a potential drug for the treatment of AD. In the preliminary project of iGEM, we did not find any project related to astragalin. Therefore, we synthesized some new gene sequences and uploaded them. The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS and UGT. Every sequence we submitted has been verified by sequencing and protein expression, and all of them can work well.

Detailed results are listed below.

Fig. 1: The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS and UGT.

     His-ATUGT + EGFP

     UGT is an enzyme catalyzing kaempferol into astragalin, and is the largest one among the three enzymes we used in our pathway (F3H, FLS, UGT), so we firstly constructed a His-ATUGT + EGFP vector to test the expression of UGT gene.

     Protocol we used:

     1. Verification of the sequence. The sequence we submitted here was come from (Arabidopsisthaliana). In order to get a better yield, we have done codon optimization for E.coli expression for this sequence. We contacted a biology company to synthesize the sequence.

     2. We constructed it into an EGFP-tagged plasmid and transformed the plasmids into E. coli BL21(DE3).

     3. After culture overnight, we picked a single colony and added it into a 4 ml LB medium with the corresponding antibiotic. The mix was then shaken at 37℃ until OD = 600.

     4. Dividing the liquid medium into 23 parts, adding 1 mM IPTG to two of them for inducing the expression 16℃ at 12 h or 36 h. The other one added nothing to serve as a control.

     5. Centrifuged the bacterial solution at 12000 g, and the precipitation was mixed with RIPA as a lysis buffer. Added loading buffer to heat at 96℃ for 10 min, underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.

     According to our SDS-PAGE result, we could see a band at around 52.04kD, which is in line with the molecular weight of UGT. This band only occurred in the IPTG group, which confirmed our induction. Besides, UGT expression at 12 h was higher than that at 36 h. We proposed that this may be due to a large number of bacteria at 36 h and the high survival pressure, which is not conducive to protein expression. Taken together, we have successfully expressed UGT in E.coli.

Fig. 2: plasmid to express atUGT

Fig. 3: SDS-page for atUGT expression

Fig. 4: HPLC results of the product reactions by AtF3H, AtFLS and AtUGT

This part and the other biological part K4236100 that expresses the AtF3H and AtFLS enzymes were expressed overnight in E. coli by iPTG induction. Naringenin was then added to the bacterial culture and reacted for 24 hours. The supernatant was extracted from the final product for HPLC measurement. Results show successful synthesis of astragalin according to comparison with the standard sample. The result indicates AtUGT can successfully express and convert kaempferol into naringenin.

Other Information

Sequence and Features