Plasmid

Part:BBa_K4516019

Designed by: Kong Yangyang   Group: iGEM22_Jiangsu_United   (2022-08-14)
Revision as of 07:31, 26 September 2022 by Cxl1102 (Talk | contribs)


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.

Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1..

hFoxO1-F.luciferase-R.luciferase：a platform to screen a small molecule FoxO1 antagonist

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 783
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4363
    Illegal SapI.rc site found at 3273


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