Difference between revisions of "Part:BBa K4414010"
Line 3: Line 3:
 
<partinfo>BBa_K4414010 short</partinfo>
 
<partinfo>BBa_K4414010 short</partinfo>
  
−
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)
+
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==
 
==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)
+
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).
 
<html>
 
<html>
  
Line 41: Line 41:
  
 
===Method===
 
===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)
+
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).
 
<html>
 
<html>
  

Revision as of 17:40, 11 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).

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