Part:BBa_K4236007

This is a part to code proteins F3H-FLS+UGT

The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS and UGT. We want to construct a big plasmid consisting of three genes we have introduced to produce astragalin. In order to better express each enzyme, we tried to connect the smaller F3H and FLS with a linker to form a fusion protein, and the larger UGT was expressed separately. Therefore, AtF3H-AtFLS + AtUGT expression vector was constructed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

     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.

     AtF3H-AtFLS+AtUGT

     The final try is to construct a big plasmid consisting of three genes we have introduced to produce astragalin. In order to better express each enzyme, we tried to connect the smaller F3H and FLS with a linker to form a fusion protein, and the larger UGT was expressed separately. Therefore, CSF3H-CuFLS +AtUGT expression vector was constructed.

    Protocol we used:

    1. We constructed it into a pETDuet plasmid and transformed the plasmids into E. coli BL21(DE3).

    2. 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.

    3. 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.

    4. 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 two bands at around 81 kD and 51kD, which are in line with the molecular weight of F3H-FLS fusion protein and UGT. The two bands only occurred in the IPTG group, which confirmed our induction. in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction was slightly higher than that with 36 h induction. Taken together, we have successfully expressed the whole pathway to produce astragalin in E.coli.

Fig. 2: Plasmid to express the whole pathway

Fig. 3: SDS-page for the whole pathway expression

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

This part constructs the whole pathway to convert naringenin into astragalin. The genes 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 the part functions as expected.