Difference between revisions of "Part:BBa K4414010"

 
 
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<partinfo>BBa_K4414010 short</partinfo>
 
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This basic part consists of the amino-terminal domain (NTD), DNA binding domain (DBD), hinge region and ligand binding domain (LBD)([[Part:BBa_K4414000]])(Weikum, Knuesel, Ortlund, & Yamamoto, 2017).
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==Usage and Biology==
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As glucocorticoid receptor, it can function both as a transcription factor that binds to glucocorticoid response elements (GRE) in the promoters of glucocorticoid responsive genes to activate their transcription, and as a regulator of other transcription factors. This receptor is typically found in the cytoplasm, but upon ligand binding, is transported into the nucleus(Lu & Cidlowski, 2005). We obtained this DNA by PCR amplification from human cDNA.This part can be used in level 1 biological laboratory.
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Figure1. Schematic figure of BBa_K4414010.
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4414010 SequenceAndFeatures</partinfo>
 
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<partinfo>BBa_K4414010 parameters</partinfo>
 
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==Functional Validation==
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===Method===
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1. HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414010and SEAP with GRE3 or GRE6. 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 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol(Shao, Qiu, & Xie, 2021).
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Figure 2: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-Tyr into cells
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2. HEK-293T cells were transfected with plasmid encoding BBa_K4414010 and ([[Part:BBa_K1123017]]). Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. The fluorescence intensity of cells was observed 24 h after posting glucocorticoids treatment.
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===Result===
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Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1-2 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 2). The fluorescence microscopic image showed GR locates in the nucleus whether treated with glucocorticoids or not (Figure 3).
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<figure class="figure">
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<img src="https://static.igem.wiki/teams/4414/wiki/10-4.png" class="figure-img img-fluid rounded"  height="350px">
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Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.
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<img src="https://static.igem.wiki/teams/4414/wiki/10-5.png" class="figure-img img-fluid rounded"  height="350px">
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Figure4. Fluorescence intensity of cells mediated by BBa_K4414010 and ([[Part:BBa_K1123017]]).
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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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2. Lu, N. Z., & Cidlowski, J. A. (2005). Translational Regulatory Mechanisms Generate N-Terminal Glucocorticoid Receptor Isoforms with Unique Transcriptional Target Genes. Molecular Cell, 18(3), 331-342. doi:https://doi.org/10.1016/j.molcel.2005.03.025
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3.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 03:53, 14 October 2022


NR3C1

This basic part consists of the amino-terminal domain (NTD), DNA binding domain (DBD), hinge region and ligand binding domain (LBD)(Part:BBa_K4414000)(Weikum, Knuesel, Ortlund, & Yamamoto, 2017).

Usage and Biology

As glucocorticoid receptor, it can function both as a transcription factor that binds to glucocorticoid response elements (GRE) in the promoters of glucocorticoid responsive genes to activate their transcription, and as a regulator of other transcription factors. This receptor is typically found in the cytoplasm, but upon ligand binding, is transported into the nucleus(Lu & Cidlowski, 2005). We obtained this DNA by PCR amplification from human cDNA.This part can be used in level 1 biological laboratory.

Figure1. Schematic figure of BBa_K4414010.


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

Method

1. HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414010and SEAP with GRE3 or GRE6. 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 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol(Shao, Qiu, & Xie, 2021).

Figure 2: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-Tyr into cells


2. HEK-293T cells were transfected with plasmid encoding BBa_K4414010 and (Part:BBa_K1123017). Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. The fluorescence intensity of cells was observed 24 h after posting glucocorticoids treatment.


Result

Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1-2 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 2). The fluorescence microscopic image showed GR locates in the nucleus whether treated with glucocorticoids or not (Figure 3).

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.
Figure4. Fluorescence intensity of cells mediated by BBa_K4414010 and (Part:BBa_K1123017).

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. Lu, N. Z., & Cidlowski, J. A. (2005). Translational Regulatory Mechanisms Generate N-Terminal Glucocorticoid Receptor Isoforms with Unique Transcriptional Target Genes. Molecular Cell, 18(3), 331-342. doi:https://doi.org/10.1016/j.molcel.2005.03.025

3.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