Difference between revisions of "Part:BBa K4414024"
 
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<partinfo>BBa_K4414024 short</partinfo>
 
<partinfo>BBa_K4414024 short</partinfo>
  
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This composite part consists of an N-terminal tetR(BBa_K4414009) domain and a C-terminal NR3C1 LBD(BBa_K4414000) domain fused with a GGGSG linker. It is designed to sense glucocorticoids and activates the transcription of the reporter gene.
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This composite part consists of an N-terminal tetR([[Part:BBa_K4414009]]) domain and a C-terminal GR LBD([[Part:BBa_K4414000]]) domain fused with a GGGSG linker. 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 C 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]
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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 C 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 et al., 2017).
  
 
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<figure class="figure">
 
<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/1/17/T--NUDT_CHINA--Part_PixD-PixE_Schematic.png" class="figure-img img-fluid rounded"  height="250px">
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<img src="https://static.igem.wiki/teams/4414/wiki/24-1.png" class="figure-img img-fluid rounded"  height="350px">
  
 
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Figure1. Schematic figure of BBa_K4414024 and BBa_K4414041
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Figure1. Schematic figure of BBa_K4414024 and ([[Part:BBa_K4414041]])
  
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4414024 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4414024 SequenceAndFeatures</partinfo>
  
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==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 BBa_K4414024 and TCE-SEAP([[Part:BBa_K4414041]]).
 
===Method===
 
===Method===
 
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Cells were treated with 10, 50, or 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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<figure class="figure">
 
<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/6/65/T--NUDT_CHINA--Part_Validation_SEAP_PixE-PixD.png
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<img src="https://static.igem.wiki/teams/4414/wiki/24-2.jpg
 
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HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414024 and TCE-SEAP(BBa_K4414041). Cells were treated with 10, 50, or 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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Figure2.Schematic representation of the experimental process of validation for BBa_K4414024 and ([[Part:BBa_K4414041]]).
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===Result===
 
===Result===
 
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Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (2-5 folds). A dose dependence was observed within 0-50 nM of glucocorticoid (Figure 3).
  
 
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<img src="https://2021.igem.org/wiki/images/d/d6/T--NUDT_CHINA--Part_Result_00-01_.png
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<img src="https://static.igem.wiki/teams/4414/wiki/24-3.png
 
" class="figure-img img-fluid rounded"  height="350px">
 
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Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (2-5 folds). A dose dependence was observed within 0-50 nM of glucocorticoid (Figure 2).
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</figure>
 
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Figure2. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414024.
 
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Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414024.
  
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==Reference==
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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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[1] Dine E, Gil AA, Uribe G, Brangwynne CP, Toettcher JE. Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli. Cell Syst. 2018 Jun 27;6(6):655-663.e5. doi: 10.1016/j.cels.2018.05.002. Epub 2018 May 30. PMID: 29859829; PMCID: PMC6023
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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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[2] Yuan H, Bauer CE. PixE promotes dark oligomerization of the BLUF photoreceptor PixD. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11715-9. doi: 10.1073/pnas.0802149105. Epub 2008 Aug 11. PMID: 18695243; PMCID: PMC2575306.
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Latest revision as of 17:49, 11 October 2022


tetR-GGGSG-LBD

This composite part consists of an N-terminal tetR(Part:BBa_K4414009) domain and a C-terminal GR LBD(Part:BBa_K4414000) domain fused with a GGGSG linker. 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 C 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 et al., 2017).

Figure1. Schematic figure of BBa_K4414024 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 BBa_K4414024 and TCE-SEAP(Part:BBa_K4414041).

Method

Cells were treated with 10, 50, or 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).

Figure2.Schematic representation of the experimental process of validation for BBa_K4414024 and (Part:BBa_K4414041).

Result

Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (2-5 folds). A dose dependence was observed within 0-50 nM of glucocorticoid (Figure 3).

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414024.

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