Difference between revisions of "Part:BBa K3522008"

 
 
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<partinfo>BBa_K3522008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3522008 SequenceAndFeatures</partinfo>
  
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Part BBa_K35220008 is one of the basic parts of Part BBa_K3522013
  
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===Contribution===
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===Functional Parameters===
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Biology
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<partinfo>BBa_K3522008 parameters</partinfo>
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Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Its typical functional domains include DNA binding domain and ligand binding domain. After binding its ligand, FXR forms a heterodimer with another nuclear receptor retinoid X receptor, and then the receptor dimer binds to the FXR response element (FXRE) located in the promoter region of the FXR target gene, thereby, regulating the transcription of these genes.
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===Engineering Success===
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Transactivation of FXRE by FXR-RXRα complex
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Part BBa_K3522013 consists of a FXRE fragment and luciferase gene. When presence of FXR and RXRα, the FXRE is activated, subsequently, the luciferase gene is transcribed. Such a transactivation assay cam be used to verify the antagonistic effect of H7 on FXR.
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Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.
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[[File:T--NOFLS_YZ-BBa_K3522013 Fig 1.jpg|500px|thumb|center|Figure 1]]
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                            Figure 1. H7 as an FXR antagonist inhibited transactivation activity.
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HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064: FXR agonist, GS: FXR antagonist. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
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==Improvement==
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In 2010, the team iGEM10_Debrecen-Hungary designed a part: BBa_K364201 to fulfill the function of a Nuclear Receptor Tookit., which is the coding sequence of human RXR DBD. Although they have successfully constructed the toolkit, they have not used it in solving specific problems related with human health.
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Based on their achievement, our team iGEM20_XDFYZ (NOFLS_YZ) designed a new part BBa_K3522008, whose sequence varies from that of the existing part BBa_K364201 (Fig3) in length and organization.
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[[File:T--NOFLS_YZ improve.png|500px|thumb|center|Figure 3]]
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                        Fig3. The difference of part BBa_K3522008 and BBa_K364201’s sequence.
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We use this new part to construct a platform to screen potential small molecular compounds for Type 2 diabetes mellitus. Besides part BBa_K3522008, our platform needs other parts to co-transfect into mammal cell HEK293T (please see our composite part BBa_K3522013). The chimeric receptor FXRa can interact with RXRa. The formation of RXR-heterodimers can be decreased by adding FXRa-agonist. And then the luciferase expression is few. Finally, we can successfully screen compounds FXRa-agonist by detecting the value of luciferase.
 +
After testing the function of our parts, we have successfully screened a compound named H7. It suggests our engineering platform should be functional and may have significant meaning in screening drugs for Type 2 diabetes mellitus

Latest revision as of 17:12, 27 October 2020


RXRα

BBa_K3522008 is a protein domain of retinoid X receptor α (RXRα), which forms a heterodimer with farnesoid X receptor α (FXRα) to modulate the expression of its target genes.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 705
    Illegal BamHI site found at 858
    Illegal BamHI site found at 972
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1270
    Illegal AgeI site found at 558
  • 1000
    COMPATIBLE WITH RFC[1000]

Part BBa_K35220008 is one of the basic parts of Part BBa_K3522013

Contribution

Biology Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Its typical functional domains include DNA binding domain and ligand binding domain. After binding its ligand, FXR forms a heterodimer with another nuclear receptor retinoid X receptor, and then the receptor dimer binds to the FXR response element (FXRE) located in the promoter region of the FXR target gene, thereby, regulating the transcription of these genes.

Engineering Success

Transactivation of FXRE by FXR-RXRα complex Part BBa_K3522013 consists of a FXRE fragment and luciferase gene. When presence of FXR and RXRα, the FXRE is activated, subsequently, the luciferase gene is transcribed. Such a transactivation assay cam be used to verify the antagonistic effect of H7 on FXR. Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.

Figure 1
                            Figure 1. H7 as an FXR antagonist inhibited transactivation activity.

HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064: FXR agonist, GS: FXR antagonist. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

Improvement

In 2010, the team iGEM10_Debrecen-Hungary designed a part: BBa_K364201 to fulfill the function of a Nuclear Receptor Tookit., which is the coding sequence of human RXR DBD. Although they have successfully constructed the toolkit, they have not used it in solving specific problems related with human health.

Based on their achievement, our team iGEM20_XDFYZ (NOFLS_YZ) designed a new part BBa_K3522008, whose sequence varies from that of the existing part BBa_K364201 (Fig3) in length and organization.

Figure 3
                        Fig3. The difference of part BBa_K3522008 and BBa_K364201’s sequence.

We use this new part to construct a platform to screen potential small molecular compounds for Type 2 diabetes mellitus. Besides part BBa_K3522008, our platform needs other parts to co-transfect into mammal cell HEK293T (please see our composite part BBa_K3522013). The chimeric receptor FXRa can interact with RXRa. The formation of RXR-heterodimers can be decreased by adding FXRa-agonist. And then the luciferase expression is few. Finally, we can successfully screen compounds FXRa-agonist by detecting the value of luciferase. After testing the function of our parts, we have successfully screened a compound named H7. It suggests our engineering platform should be functional and may have significant meaning in screening drugs for Type 2 diabetes mellitus