Difference between revisions of "Part:BBa K4414025"
 
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==Usage and Biology==
 
==Usage and Biology==
 
As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription.
 
As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription.
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The NR3C1 LBD domain on the N terminus is the ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression.[1] GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. Tet R in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. NLS (nuclear localization signal) helps the nucleophilic proteins better move into the nucleus. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 
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The GR LBD domain on the N terminus is the ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression(Weikum et al., 2017). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. Tet R in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter ([[Part:BBa_K4016011]]) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. NLS (nuclear localization signal) helps the nucleophilic proteins better move into the nucleus. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 
  
  
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<figure class="figure">
 
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<img src="https://static.igem.org/mediawiki/parts/5/5b/T--NUDT_CHINA--Part_SchematicFigure_15_.png" class="figure-img img-fluid rounded"  height="350px">
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<img src="https://static.igem.wiki/teams/4414/wiki/25-1.png" class="figure-img img-fluid rounded"  height="350px">
  
 
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Figure 1. Schematic figure of BBa_K4414025 and BBa_K4414041.
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Figure 1. Schematic figure of BBa_K4414025 and ([[Part:BBa_K4414041]]).
  
  
  
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===Sequecing===
 
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The plasmid was sequenced correct.
 
  
 
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==Functional test==
 
==Functional test==
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To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64(BBa_K4414025) and TCE-SEAP(BBa_K4414041).  
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To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64 (BBa_K4414025) and TCE-SEAP([[Part:BBa_K4414041]]).  
 
===Method===
 
===Method===
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Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol.[2]
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Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol(Shao, Qiu, & Xie, 2021).
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<figure class="figure">
 
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<img src="https://static.igem.org/mediawiki/parts/6/61/T--NUDT_CHINA--Part_Validation_CCK-8_72h.png
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<img src="https://static.igem.wiki/teams/4414/wiki/25-2.png
 
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Figure2.Schematic representation of the experimental process of validation for BBa_K4414025 and BBa_K4414041.
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Figure2.Schematic representation of the experimental process of validation
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===Result===
 
===Result===
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Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds).  
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Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds).
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<figure class="figure">
 
<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/e/e5/T--NUDT_CHINA--Part_Result_15_.png
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<img src="https://static.igem.wiki/teams/4414/wiki/25-3.png" class="figure-img img-fluid rounded"  height="350px">
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Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414025.  
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Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414025.
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Compared with the experimental results of scFv_cyclinE1-Coh2, the results of Coh2-scFv_cyclinE1 had more obvious changes than the control group, which showed that the design of Coh2-scFv_cyclinE1 was more effective for our system.
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==Reference==
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1. Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol, 18(3), 159-174. doi:10.1038/nrm.2016.152
  
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===Reference===
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2. Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol, 2312, 35-57. doi:10.1007/978-1-0716-1441-9_3
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[1].Weikum ER, Knuesel MT, Ortlund EA, Yamamoto KR. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol. 2017 Mar;18(3):159-174. doi: 10.1038/nrm.2016.152. Epub 2017 Jan 5. PMID: 28053348; PMCID: PMC6257982.
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[2].Shao J, Qiu X, Xie M. Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol. 2021;2312:35-57. doi: 10.1007/978-1-0716-1441-9_3. PMID: 34228283.
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Latest revision as of 18:21, 11 October 2022


LBD-GGGGGSG-tetR-GGGSG--NLS-vp64

This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.


Usage and Biology

As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription. The GR LBD domain on the N terminus is the ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression(Weikum et al., 2017). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. Tet R in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (Part:BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. NLS (nuclear localization signal) helps the nucleophilic proteins better move into the nucleus. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 



Figure 1. Schematic figure of BBa_K4414025 and (Part:BBa_K4414041).



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional test

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64 (BBa_K4414025) and TCE-SEAP(Part:BBa_K4414041).

Method

Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol(Shao, Qiu, & Xie, 2021).

Figure2.Schematic representation of the experimental process of validation

Result

Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds).

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414025.


Reference

1. Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol, 18(3), 159-174. doi:10.1038/nrm.2016.152

2. Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol, 2312, 35-57. doi:10.1007/978-1-0716-1441-9_3