Difference between revisions of "Part:BBa K4414000"

 
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
 +
 
<partinfo>BBa_K4414000 short</partinfo>
 
<partinfo>BBa_K4414000 short</partinfo>
  
−
As an intelligent cell that senses stress, the most critical step is to detect and respond to changes in stress hormones. We choose cortisol as the detection object.NR3C1 is a cortisol receptor gene, and we only truncated its LBD in response to changes in cortisol concentration.
+
This part is the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR)..
 +
 
 +
==Usage and Biology==
 +
 
 +
The human glucocorticoid receptor is encoded by NR3C1 (Nuclear Receptor Subfamily 3, Group C, Member 1) gene. GR functions as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription (Lu & Cidlowski, 2005). In the absence of glucocorticoids, GR binds to small molecular chaperone proteins in the cytoplasm and stays in the cytoplasm. Following glucocorticoid stimulation, it changes its conformation and dissociates from the chaperone protein, thereby exposing the nuclear localization sequence on the ligand-binding domain, which facilitates the entry of the GR into the nucleus.  
 +
GR consists of an N-terminus binding domain (NTD), a DNA binding domain (DBD), and a ligand binding domain (LBD). As the major functional domain, the LBD domain is responsible for sensing glucocorticoid signals and translocating the protein into the nucleus (Monsalve et al., 2019). It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression (Weikum et al., 2017).
 +
 
 +
 
 +
<html>
 +
 
 +
<figure class="figure">
 +
<img src="https://static.igem.wiki/teams/4414/wiki/000-1.jpg" class="figure-img img-fluid rounded"  height="200px">
 +
 
 +
</figure>
 +
 
 +
</html>
 +
Figure1： Linear domain structure of glucocorticoid receptor (GR) (Weikum et al., 2017)
 +
 
 +
===Sequecing===
 +
The plasmid was sequenced correct.
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
===Sequence and Features===
 
<partinfo>BBa_K4414000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4414000 SequenceAndFeatures</partinfo>
  
Line 17: Line 35:
 
<partinfo>BBa_K4414000 parameters</partinfo>
 
<partinfo>BBa_K4414000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
===Functional characterization and result===
 +
 +
We constructed a plasmid that linked LBD to the fluorescent protein EGFP to verify the function of LBD. Cells are treated with 500 and 1000 nM glucocorticoids 6 h after transfection of plasmids into HEK-293T cells. Cells without glucocorticoid treatment were used as control. The results showed that glucocorticoid-treated cells fluorescence was mainly present in the nucleus compared to non-treated control, suggesting that glucocorticoids can bind to LBD and enter the nucleus.
 +
<html>
 +
 +
<figure class="figure">
 +
<img src="https://static.igem.wiki/teams/4414/wiki/000-2.jpg" class="figure-img img-fluid rounded"  height="250px">
 +
 +
</figure>
 +
 +
</html>
 +
 +
===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.

Revision as of 07:17, 9 October 2022


Ligand-binding domain (LBD) of NR3C1

This part is the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR)..

Usage and Biology

The human glucocorticoid receptor is encoded by NR3C1 (Nuclear Receptor Subfamily 3, Group C, Member 1) gene. GR functions as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription (Lu & Cidlowski, 2005). In the absence of glucocorticoids, GR binds to small molecular chaperone proteins in the cytoplasm and stays in the cytoplasm. Following glucocorticoid stimulation, it changes its conformation and dissociates from the chaperone protein, thereby exposing the nuclear localization sequence on the ligand-binding domain, which facilitates the entry of the GR into the nucleus. GR consists of an N-terminus binding domain (NTD), a DNA binding domain (DBD), and a ligand binding domain (LBD). As the major functional domain, the LBD domain is responsible for sensing glucocorticoid signals and translocating the protein into the nucleus (Monsalve et al., 2019). 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： Linear domain structure of glucocorticoid receptor (GR) (Weikum et al., 2017)

Sequecing

The plasmid was sequenced correct.


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 characterization and result

We constructed a plasmid that linked LBD to the fluorescent protein EGFP to verify the function of LBD. Cells are treated with 500 and 1000 nM glucocorticoids 6 h after transfection of plasmids into HEK-293T cells. Cells without glucocorticoid treatment were used as control. The results showed that glucocorticoid-treated cells fluorescence was mainly present in the nucleus compared to non-treated control, suggesting that glucocorticoids can bind to LBD and enter the nucleus.

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.