Difference between revisions of "Part:BBa K4516001"
| Line 6: | Line 6: | ||
== Profile== | == Profile== | ||
Name: hFoxO1 | Name: hFoxO1 | ||
| + | |||
Origin: HepG2 cell genome | Origin: HepG2 cell genome | ||
| Line 14: | Line 15: | ||
== Engineering Success== | == Engineering Success== | ||
How we design our plasmid | 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. | 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. | ||
[[File:T--Jiangsu United--BBa K4516019-figure1.png|500px|thumb|center|Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1..]] | [[File:T--Jiangsu United--BBa K4516019-figure1.png|500px|thumb|center|Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1..]] | ||
How we build our plasmid | 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. | 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. | ||
[[File:T--Jiangsu United--BBa K4516019-figure2.png|500px|thumb|center|Fig.2 Agarose gel electrophoresis of PCR product. | [[File:T--Jiangsu United--BBa K4516019-figure2.png|500px|thumb|center|Fig.2 Agarose gel electrophoresis of PCR product. | ||
Revision as of 09:10, 26 September 2022
hFoxO1
hFoxO1
Profile
Name: hFoxO1
Origin: HepG2 cell genome
Contribution
Forkhead box protein (Fox) is a family of transcription factors. The DNA binding region of this family of proteins has a conserved winglike helix structure. There are currently 17 subfamilies in this family, of which the FoxO subfamily is the most well studied. There are four subtypes: FoxO1, FoxO3, FoxO4, and FoxO6 in mammals. FoxO1 has four domains, which are DNA-binding domain, nuclear localization domain, nuclear export sequence, and transcriptional activation domain. It binds with IRE sequence and plays a role in regulating downstream genes. FoxO1 is mainly expressed in insulin-responsive tissues, The main role of FoxO1 is to regulate downstream target genes, such as PEPCK, G6Pase, PGC1-α, and PDK-4 to promote gluconeogenesis and can regulate cell proliferation, gluconeogenesis, and energy metabolism.
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) 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.3). 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 289
Illegal NotI site found at 298 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 554
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]