Part:BBa_K4255100
pRSFDuet-gbF3H
F3H: catalyze flavanone into dihydrokaempferol
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 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.
pETDUET- F3H
The first new part we uploaded this year is to express F3H, which is enzyme working to catalyze dihydrokaempferol into dihydromyricetin. The sequence we submitted here was come from Ginkgo biloba L. And we has done codon optimization for E.coli expression.
Major protocol we use:
We asked biological company to synthesize the sequence at first. And we constructed it into pRSFDUET 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 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 ~43kD, 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 F3H gene.
We also studied the effect of induction time on the expression level, and set the induction time as 16 h, 20 h and 24 h at 16℃. Three parallel experiments were performed at each induction time. There was an obvious expression band (shown by red arrow), but the expression levels were similar at the induction time of 16h, 20h and 24h.
Besides of time, we do concern whether expression temperature could make a difference. In our next experiment, the induction temperatures were set to be 16℃, 20℃, 30℃ and 37℃, respectively. There are also three repeats for each induced temperature. The result told us the expression level was higher at 20℃.
Based on the result before, we thought about if the concentration of IPTG could effect the expression level, so we set 0.05mM、0.1mM、0.2mM、0.5mM、1mM、0.2mM concentration to give a test.
Other Information
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1184
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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