Difference between revisions of "Part:BBa K4255102"

Revision as of 11:28, 12 October 2022

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

We expect a protein band around 95kD on the SDS-Page results. The above SDS-Page was carried out at different inoculation temperatures after iPTG induction. The band above (greater than) 100kD is not any protein of our design and should be disregarded. There is vague expression clues at 20 and 30 degree Celsius.

Figure 4. Quantitative analysis of the above SDS-Page results.

We digitized the photo by reading the grey scale of each protein band. The numbers clearly show an increased expression at 20 and 30 degree C but the expression drops to background level at high temperature.

Figure 5. Product in the bacteria culture after induction and inoculation. The product changed color remarkably with varying pH.

Strongest evidence of the successful production of the four enzymes is shown in the product bacteria culture. After iPTG induction, we added the precursor into the culture and incubate for 72 hours. The product culture showed a pink-ish purple, indicating the presence of delphinidin. All intermediate substance along the reaction pathway is colorless. The product solution also drastically changed this color upon tuning the pH, an unique feature of delphinidin.

Other Information

Sequence and Features