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This year our project is focused on the synthesis of anthocyanin, which has a lot of research and reporting on this canonical pathway. But due to anthocyanins are ubiquitous in plants, therefore, each enzyme can find multiple homologous proteins. To get the better yeild, we first compared 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.

Fig. 1: Metabolism pathway for anthocyanin synthesis

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 4CL, CHS and CHI sequences come from (三个物种). We constructed the first three enzymes on the pETDUET vector, in which 4CL was linked to CHS via linker to form a fusion protein and CHI was expressed independently. The function of the enzymes can be listed below: 4CL: catalyze phenylalanine into 4-coumaryl coenzyme A CHS: catalyze 4-coumaryl coenzyme A into chalcone CHI: catalyze chalcone into flavanone

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 1 mM IPTG for induction and shook at 16℃ 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 (请老师补充分子量大小)kD, 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 and CHS, and an individual protein of CHI. (需要补充载体设计图与表达结果图)

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