Difference between revisions of "Part:BBa K4414010"
Line 34: Line 34:
  
 
===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]
−
To test the feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene and a plasmid with TCE-Tyr into HEK-293T cells. Cells were treated with 10, 50, or 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. An excitation wave of 554nm was used to make the protein visible under a fluorescence microscope. Then we can observe the red fluorescence of cells at 24 h post glucocorticoids treatment.
+
  
 
<html>
 
<html>
Line 47: Line 46:
 
Figure 3: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-Tyr into cells
 
Figure 3: 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.
 +
<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===
−
The test results are as follows:
+
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).
 
<html>
 
<html>
  
Line 58: Line 67:
  
 
</html>
 
</html>
−
Figure 4: The red fluorescence of HEK-293T cells at 24 h  in the case of 0 and 100 glucocorticoid stimulation.
+
Figure2. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.
 +
<html>
  
−
As it turns out, our pathway is feasible, and we visualized pressure changes that are invisible and difficult to self-accurately assess, characterizing whether there is pressure and the magnitude of pressure in terms of red fluorescence production and strength grade. Future iGEM teams can use this part by simply replace the upstream part to transform the abstract into concrete work as we did.
+
<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>
 +
Figure3. Fluorescence intensity of cells mediated by BBa_K4414010 and 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]Syverud BC, Gumucio JP, Rodriguez BL, Wroblewski OM, Florida SE, Mendias CL, Larkin LM. A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications. Tissue Eng Part C Methods. 2018 May;24(5):263-271. doi: 10.1089/ten.TEC.2017.0406. Epub 2018 Apr 10. PMID: 29490563; PMCID: PMC5946766.  
+
[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
 +
 
 +
[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]Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004 Dec;22(12):1567-72. doi: 10.1038/nbt1037. Epub 2004 Nov 21. PMID: 15558047.
+
[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.

Revision as of 06:26, 9 October 2022


NR3C1

This basic part consists of the amino-terminal domain (NTD), DNA binding domain (DBD), hinge region and ligand binding domain (LBD)(BBa_K4414000).[1]

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]

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_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]

Figure 3: 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.

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).

Figure2. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414010.
Figure3. Fluorescence intensity of cells mediated by BBa_K4414010 and BBa_K1123017.

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

[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.

[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.