Difference between revisions of "Part:BBa K4516001"
 
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hFoxO1
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== Profile==
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Name: hFoxO1
  
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Origin: HepG2 cell genome
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== Contribution==
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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.
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== Engineering Success==
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How we design our plasmid
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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.
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[[File:T--Jiangsu United--BBa K4516019-figure1.png|500px|thumb|center|Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1..]]
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How we build our plasmid
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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.
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[[File:T--Jiangsu United--BBa K4516019-figure2.png|500px|thumb|center|Fig.2 Agarose gel electrophoresis of PCR product.
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(A)Lane 1 is the hFoxO1 target band...]]
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a) Protein expression and verification
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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.
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[[File:T--Jiangsu United--BBa K4516019-figure4.png|500px|thumb|center|Fig.3 Protein determination of HepG2 cells after transfection..]]
  
 
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<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
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Latest revision as of 10:29, 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.

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

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.

Fig.2 Agarose gel electrophoresis of PCR product. (A)Lane 1 is the hFoxO1 target band...

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.

Fig.3 Protein determination of HepG2 cells after transfection..

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 289
    Illegal NotI site found at 298
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 554
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]