Difference between revisions of "Part:BBa K4414037"
 
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<partinfo>BBa_K4414037 short</partinfo>
 
<partinfo>BBa_K4414037 short</partinfo>
  
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This composite part consists of a C-terminal tetR(BBa_K4414009) domain and an NR3C1 LBD(BBa_K4414000) domain fused with NES(BBa_K4414003). It is designed to sense glucocorticoids and activates the transcription of the reporter gene.
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This composite part consists of a C-terminal tetR([[Part:BBa_K4414009]]) domain and an GR LBD([[Part:BBa_K4414000]]) domain fused with NES([[Part:BBa_K4414003]]). It is designed to sense glucocorticoids and activates the transcription of the reporter gene.
  
 
==Usage and Biology==
 
==Usage and Biology==
  
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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. This part consists of a tetR DNA binding domain, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The NR3C1 LBD domain on the N terminal 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] NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .
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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. This part consists of a tetR DNA binding domain, which binds to the TCE promoter ([[Part:BBa_K4016011]]) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The GR LBD domain on the N terminal 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, Knuesel, Ortlund, & Yamamoto, 2017). NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .
  
  
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<figure class="figure">
 
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<img src="https://static.igem.wiki/teams/4414/wiki/037-1.png" class="figure-img img-fluid rounded"  height="550px">
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<img src="https://static.igem.wiki/teams/4414/wiki/037-1.png" class="figure-img img-fluid rounded"  height="350px">
  
 
</figure>
 
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</html>
  
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Figure1. Figure1. Schematic figure of BBa_K4414037 and BBa_K4414041
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Figure1. Figure1. Schematic figure of BBa_K4414037 and ([[Part:BBa_K4414041]])
  
  
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==Functional Validation==
 
==Functional Validation==
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HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 and TCE-SEAP([[Part:BBa_K4414041]]).
  
 
===Method===
 
===Method===
  
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HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 and TCE-SEAP. Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2]
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Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol (Shao, Qiu, & Xie, 2021).
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<html>
 
<html>
  
 
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<figure class="figure">
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<img src="https://static.igem.wiki/teams/4414/wiki/37-2.png" class="figure-img img-fluid rounded"  height="450px">
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<img src="https://static.igem.wiki/teams/4414/wiki/37-2.png" class="figure-img img-fluid rounded"  height="350px">
  
 
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Figure 2
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Figure 2:Cotransfected our plasmid with the  TCE-SEAP into cells
  
  
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<img src="https://static.igem.wiki/teams/4414/wiki/NUDT2022-037-3.png" class="figure-img img-fluid rounded"  height="650px">
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<img src="https://static.igem.wiki/teams/4414/wiki/nudt2022-037-3.png" class="figure-img img-fluid rounded"  height="350px">
  
 
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Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.
 
Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.
  
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Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control. A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).
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Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.7-2.0 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).
  
 
===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 17:55, 11 October 2022


TetR-GSG-NES-GSG-LBD

This composite part consists of a C-terminal tetR(Part:BBa_K4414009) domain and an GR LBD(Part:BBa_K4414000) domain fused with NES(Part:BBa_K4414003). It is designed to sense glucocorticoids 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. This part consists of a tetR DNA binding domain, which binds to the TCE promoter (Part:BBa_K4016011) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The GR LBD domain on the N terminal 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, Knuesel, Ortlund, & Yamamoto, 2017). NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .



Figure1. Figure1. Schematic figure of BBa_K4414037 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 Validation

HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 and TCE-SEAP(Part:BBa_K4414041).

Method

Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol (Shao, Qiu, & Xie, 2021).

Figure 2:Cotransfected our plasmid with the TCE-SEAP into cells


Result

The test results are as follows:

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.

Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.7-2.0 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).

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