Part:BBa_K4255101
pETDuet-gm4CL-gmCHS+gmCHI
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
Usage and Biology
Engineering success SHSBNU_China
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 yield, 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.
pETDUET-4CL, CHS-CHI
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 Glycine max. We constructed the first three enzymes on the pETDUET vector, in which 4CL was linked to CHS and CHI via linkers to form a fusion protein.
The function of the enzymes can be listed below:
4CL: catalyze phenylalanine into 4-coumaryl coenzyme
CHS: catalyze 4-coumaryl coenzyme A into chalcone
CHI: catalyze chalcone into flavanone
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:
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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3580
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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