Part:BBa_K4236100

T7-RBS-atF3H+atFLS

The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS and UGT. F3H is an enzyme of 41.36 kD, which catalyzes naringenin into dihydrokaempferol. FLS is of 39.41 kD to catalyze dihydrokaempferol into kaempferol. We constructed an intact plasmid to express AtF3H and AtFLS respectively.

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.)

    AtF3H+AtFLS

    F3H is an enzyme of 41.36 kD, which catalyzes naringenin into dihydrokaempferol. FLS is 39.41 kD to catalyze dihydrokaempferol into kaempferol. We constructed an intact plasmid to express AtF3H and AtFLS respectively.

    Protocol we used:

    1. Verification of the sequence. The sequence we submitted here was come from (Arabidopsis thaliana). In order to get a better field, 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 a pETDuet 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, 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.

   &nbspAccording to our SDS-PAGE result, we could see two band at around 41.36 kD and 39.41 kD, which are in line with the molecular weight of F3H and FLS. The two band only occurred in IPTG group, which confirmed with our induction. in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction were slightly higher than that with 36 h induction. Taken together, we have successfully expressed FSH and FLS in E.coli.

Fig. 2: Plasmid to express AtF3H+AtFLS )

Fig. 3: SDS-page for AtF3H+AtFLS expression)

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

This part and the other biological part that expresses the UGT enzyme 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 AtF3H and AtFLS can successfully express and convert naringenin into the precursor kaempferol, which is subsequently converted into naringenin.

Other Features

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]