Part:BBa_K4255102

pRSFDuet-F3H, F3'5'H-DFR, ANS

We divided the seven enzymes into two parts, 4CL, CHS and CHI catalyzed the production of naringenin one by one, and naringenin was further generated into anthocyanins in the presence of F3H, F3'5'H, DFR and ANS. The F3H, F3'5'H, DFR and ANS. We constructed the last four enzymes on the pRSFDuet vector, in which F3H was fused to F3'5'H and DFR was fused to ANS. The function of the enzymes can be listed below: F3H: catalyze flavanone into dihydrokaempferol F3’5’H: catalyze dihydrokaempferol into dihydromyricetin DFR: catalyze dihydromyricetin into delphinin colorless ANS: catalyze delphinin colorless into delphinidin

Usage and Biology

Engineering success SHSBNU_China 2022

This year our project is focused on the synthesis of delphinidin, a type of anthocyanin. There has been a lot of research and reports on this substance. Because anthocyanins are ubiquitous in plants, each enzyme on the synthesis pathway can find multiple homologous proteins. We researched the literatures and picked enzymes in different species and selected the most suitable sequence for E.coli expression, we constructed several new parts, which has been submitted this year.

Figure 1. Anthocyanin synthesis pathway

We divided the seven enzymes into two parts, 4CL, CHS and CHI catalyze the production of naringenin one by one, and naringenin was further generated into anthocyanins in the presence of F3H, F3'5'H, DFR and ANS. We constructed the last four enzymes on the pRSFDuet vector, in which F3H was fused to F3'5'H and DFR was fused to ANS. The function of the enzymes can be listed below: F3H: catalyze flavanone into dihydrokaempferol F3’5’H: catalyze dihydrokaempferol into dihydromyricetin DFR: catalyze dihydromyricetin into delphinin colorless ANS: catalyze delphinin colorless into delphinidin

    

Figure 2. Plasimid for BBa K4255102

Major protocol we use:

We asked biological company to synthesize the sequence at first. And we constructed it into pETDUET plasmid. Next, we transformed the plasmids into E. coli BL21(DE3).

After the colony has grown up on the plate, we picked a single colony by a sterile tip and added it into 4 ml LB medium with the corresponding antibiotic.

Later, we added IPTG for induction and shook at overnight. We also set a control group and didn’t add IPTG into it.

Finally, we centrifuged the bacterial solution at 12000 g, discard the supernatant, and used RIPA as a lysis buffer. we added loading buffer to the supernatant which contains the protein extract, and after heating at 96℃ for 10 min, we underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.

We could see a clear band at the position of 112kD, which only appeared in the group with IPTG. The molecular weight of this band was in line with the expectation, and it was under the condition of induction, so we believed that we had successfully expressed fusion protein combined by 4CL, CHS and CHI.

     We studied the effect of induction time on the expression level, IPTG with a final concentration of 0.2 mM was added and induced at 16℃ for 16 h, 20 h and 24 h, respectively. Three repeats for each induction time. Under proper IPTG concentration induced for different times, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining at last.

Here are our results:

Figure 3. Expression test for BBa K4255101

     The pETDUET-4CL-CHS-CHI was expressed as we saw a clearly band shown by red arrow, but the expression level is similar between each time.

     Then we moved on to change expression temperature. The induction temperatures were set to be 16℃, 20℃, 30℃ and 37℃, respectively. There are also three repeats for each induced temperature.

Figure 4. Expression of BBa_K4255101 at different temperatures

     As shown in the figure, 20℃ seems to be the worst condition for pETDUET-4CL-CHS-CHI.

     Next, we set 0.05mM、0.1mM、0.2mM、0.5mM、1mM、0.2mM IPTG concentration to give a expression test as well.

Figure 5. expression test for BBa K4255101

Figure 6. expression test for BBa K4255101

     According to our result, there is not much difference between IPTG concentrations for pETDUET-4CL-CHS1-CHI expressing.

     So we successfully expressed three of the enzymes in anthocyanin synthesis pathway.

Other Information

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]