Difference between revisions of "Part:BBa K4414010"
 
(15 intermediate revisions by 4 users not shown)
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)(BBa_K4414000).[1]
+
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.[2]
+
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.
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
−
<img src="https://static.igem.wiki/teams/4414/wiki/016-1.png" class="figure-img img-fluid rounded"  height="150px">
+
<img src="https://static.igem.wiki/teams/4414/wiki/10-1.png" class="figure-img img-fluid rounded"  height="350px">
  
 
</figure>
 
</figure>
  
 
</html>
 
</html>
 +
<html>
  
−
Figure1. Schematic figure of BBa_K4414010
+
<figure class="figure">
 +
<img src="https://static.igem.wiki/teams/4414/wiki/10-2.png" class="figure-img img-fluid rounded"  height="300px">
 +
 
 +
</figure>
 +
 
 +
</html>
 +
Figure1. Schematic figure of BBa_K4414010.
  
  
Line 22: Line 29:
 
<!-- -->
 
<!-- -->
 
===Sequence and Features===
 
===Sequence and Features===
−
<partinfo>BBa_K4414016 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4414010 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
−
<partinfo>BBa_K4414016 parameters</partinfo>
+
<partinfo>BBa_K4414010 parameters</partinfo>
 
<!-- -->
 
<!-- -->
−
 
  
 
==Functional Validation==
 
==Functional Validation==
  
 
===Method===
 
===Method===
−
1. HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414010 and 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. [3]
+
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>
  
 
<figure class="figure">
 
<figure class="figure">
−
<img src="https://static.igem.wiki/teams/4414/wiki/16-3.png" class="figure-img img-fluid rounded"  height="450px">
+
<img src="https://static.igem.wiki/teams/4414/wiki/10-3.png" class="figure-img img-fluid rounded"  height="350px">
  
 
</figure>
 
</figure>
  
 
</html>
 
</html>
−
Figure 3: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-Tyr into cells
+
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 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.
+
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.
−
<html>
+
  
−
<figure class="figure">
 
−
<img src="https://static.igem.wiki/teams/4414/wiki/16-3.png" class="figure-img img-fluid rounded"  height="450px">
 
−
 
−
</figure>
 
−
 
−
</html>
 
  
 
===Result===
 
===Result===
Line 62: Line 60:
  
 
<figure class="figure">
 
<figure class="figure">
−
<img src="https://static.igem.wiki/teams/4414/wiki/016-4.png" class="figure-img img-fluid rounded"  height="650px">
+
<img src="https://static.igem.wiki/teams/4414/wiki/10-4.png" class="figure-img img-fluid rounded"  height="350px">
  
 
</figure>
 
</figure>
  
 
</html>
 
</html>
−
Figure2. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.
+
Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
−
<img src="https://static.igem.wiki/teams/4414/wiki/016-4.png" class="figure-img img-fluid rounded"  height="650px">
+
<img src="https://static.igem.wiki/teams/4414/wiki/10-5.png" class="figure-img img-fluid rounded"  height="350px">
  
 
</figure>
 
</figure>
  
 
</html>
 
</html>
−
Figure3. Fluorescence intensity of cells mediated by BBa_K4414010 and BBa_K1123017.
+
Figure4. Fluorescence intensity of cells mediated by BBa_K4414010 and ([[Part:BBa_K1123017]]).
−
<html>
+
  
−
<figure class="figure">
 
−
<img src="https://static.igem.wiki/teams/4414/wiki/016-4.png" class="figure-img img-fluid rounded"  height="650px">
 
−
 
−
</figure>
 
−
 
−
</html>
 
 
===Reference===
 
===Reference===
−
[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
+
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 NZ, Cidlowski JA. Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Mol Cell. 2005 Apr 29;18(3):331-42. doi: 10.1016/j.molcel.2005.03.025. PMID: 15866175.
+
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. 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.
+
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