Part:BBa_K4516019
hFoxO1-F.luciferase-R.luciferase：a platform to screen a small molecule FoxO1 antagonist
Profile
Name: hFoxO1-F.luciferase-R.luciferase
Base Pairs: 6187 bp
Origin: HepG2 cell genome
Properties: expression plasmid that can duplicate both in E.coli and HepG2 cells
Contribution
FoxO1, as a transcription factor, plays an important role in the regulation of blood glucose balance. FoxO1 in the liver can promote the expression of key gluconeogenesis enzyme genes, and an important mechanism of insulin regulation of blood sugar is to increase the phosphorylation of FoxO1, promote its nuclear export, and then reduce its transcriptional activation activity. According to this theory, we constructed a human FoxO1 full-length plasmid, used Firefly luciferase and Renilla luciferase as a reporter gene, established a FoxO1 transcriptional activation screening platform, and used this platform to screen out active compounds that inhibit FoxO1 transcriptional activation activity.
In order to construct a FoxO1 expression plasmid that can shuttle both in E.coli and HepG2 cells, we designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector (Fig.1), and transfected the HepG2 cells with the recombinant plasmid to set up our experiment platform.
Engineering Success
How we design our plasmid
In order to construct a FoxO1 expression plasmid that can duplicate both in E.coli and HepG2 cells, we designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector (Fig.1), and transfect the HepG2 cells with the recombinant plasmid and set up our experiment platform.
How we build our plasmid
To build the plasmid, we use PCR to amplify the hFoxO1 gene from template DNA (HepG2 cell genome), and extract the target fragment (Fig.2). At the same time, we did the plasmid extraction to obtain the plasmid pcDNA3.1. The second step was double-enzyme digestion with XhoI and KpnI. The goal of digestion was to get the linearized pcDNA3.1 vector and inserted DNA fragments of hFoxO1. The third step was to ligate the inserts and linearized vector and transfer the ligation product into DH5α competent.
(A)Lane 1 is the hFoxO1 target band. We send the constructed recombinant plasmid to a sequencing company for sanger sequencing. The returned sequencing comparison results showed that the plasmid was successfully constructed (Fig.3). Then we extract the recombinant plasmid from E.coli DH5α and transfect it into HepG2 cells to express hFoxO1 proteins.
How we test our hFoxO1
a) Protein expression and verification In order to verify if hFoxO1 protein was successfully expressed in HepG2 cells which were transfected with the correct recombinant plasmid after 12 hours, we did Western blot (Fig.4). The result shows that the protein expression level of hFoxO1 increased accompanied by the increase of plasmid concentration, indicating that hFoxO1 was expressed successfully in the cell.
improvement of an existing part
Our composite component, BBa_K4516019, is a platform to screen small molecule FoxO1 antagonists that can be used to treat type 2 diabetes. As early as 2020, the iGEM20_XDFYZ team was committed to developing a drug screening platform (BBa_K3522013) for the treatment of type 2 diabetes. They found candidate drug molecules by screening FXR antagonists. Based on their work, by reading literature and consulting experts in related fields, we found that in addition to the FXR target, there are also a FoxO1 target that plays an important role in the regulation of blood glucose balance. Therefore, we further optimized the diabetes drug screening platform and constructed a new composite part BBa_K4516019 to screen more kinds of drug molecules that can be used to treat type 2 diabetes. In order to prove the function of our new composite part hFoxO1-F. luciferase-R. luciferase, we transferred the recombinant plasmid into HepG2 to establish a transcriptional activation screening platform, and screened a totally new compound 355, which has an inhibitory effect on the transcriptional activation activity of FoxO1. Then, by detecting the effect of 355 active compounds on hepatic gluconeogenesis and the mRNA levels of key enzymes in hepatic gluconeogenesis, it was further confirmed that 355 compounds have the effect of inhibiting hepatic gluconeogenesis, thereby achieving the effect of lowering blood sugar.
Results of Compounds Screening Using this Platform To regulate the expression of hFoxO1, we used small molecular compounds from the database to examine their effects on gluconeogenesis (Table1). Compound 335 was found to have a significant role in reducing glucose concentrations in HepG2 cells. Table 1. The effects of different small molecular compounds on gluconeogenesis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 518
Illegal NotI site found at 527 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 783
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4363
Illegal SapI.rc site found at 3273
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