Difference between revisions of "Part:BBa K4414040"
 
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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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Tet R on the N terminus 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. The NR3C1 LBD domain 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 1) GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 
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Tet R on the N terminus 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. The GR LBD domain 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 (Weikum et al., 2017). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 
 
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<img src="https://static.igem.org/mediawiki/parts/3/3a/T--NUDT_CHINA--Part_SchematicFigure_12-13_.png" class="figure-img img-fluid rounded"  height="350px">
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<img src="https://static.igem.wiki/teams/4414/wiki/40-1.png" class="figure-img img-fluid rounded"  height="350px">
  
 
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Figure1.Schematic figure of BBa_K4414040 and BBa_K4414041.
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Figure1.Schematic figure of BBa_K4414040 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 Tet R-LBD-1xGS linker-VP64(BBa_K4414040) 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 Tet R-LBD-1xGS linker-VP64(BBa_K4414040) 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 24h 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 24h post glucocorticoids treatment. SEAP activity was measured according to a published protocol (Shao, Qiu, & Xie, 2021).
  
 
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<img src="https://static.igem.org/mediawiki/parts/0/07/T--NUDT_CHINA--Part_Validation_Flourescent_72h.png
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Figure2.Schematic representation of the experimental process of validation for BBa_K4414040 and BBa_K4414041.
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Figure2.Schematic representation of the experimental process of validation for BBa_K4414040 and ([[Part:BBa_K4414041]]).
  
  
 
===Result===
 
===Result===
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Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.92 folds).
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Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414040.  
 
Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414040.  
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===Reference===
 
===Reference===
  
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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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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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[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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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

Latest revision as of 18:07, 11 October 2022


TetR-GGGSG-LBD-GGGSG-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. Tet R on the N terminus 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. The GR LBD domain 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 (Weikum et al., 2017). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 

Figure1.Schematic figure of BBa_K4414040 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 Tet R-LBD-1xGS linker-VP64(BBa_K4414040) 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 24h 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 for BBa_K4414040 and (Part:BBa_K4414041).


Result

Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.92 folds).

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414040.


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