Difference between revisions of "Part:BBa K4414029"

 
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
 +
 
<partinfo>BBa_K4414029 short</partinfo>
 
<partinfo>BBa_K4414029 short</partinfo>
  
−
exchange the position of EGFP and LBD to test function, validate that the glucocorticoid-stimulated entry from the cytoplasm into the nucleus can be accomplished by retaining only the LBD fragment
+
This composite part consists of an N-Terminal EGFP (BBa_K4414005) and a C-Terminal NR3C1 LBD (BBa_K4414000) domain. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.
 +
 
 +
==Usage and Biology==
 +
 
 +
We constructed a plasmid to link LBD with the fluorescent protein EGFP to verify the function of LBD. The EGFP on the N-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the C-Terminal is a ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a trans-activating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression[1].
 +
 
 +
===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_K4414029 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4414021 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
−
<partinfo>BBa_K4414029 parameters</partinfo>
+
<partinfo>BBa_K4414021 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
==Fuctional test==
 +
===Method===
 +
 +
To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding BBa_K4414031. 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 24 h or 48 h post glucocorticoids treatment. Finally, we observe the fluorescence intensity of cells.
 +
 +
===Result===
 +
 +
Fluorescence images are shown below, which indicates that glucocorticoids can bind to LBD and enter the nucleus. This provides a basic direction of thinking for our experiments.
 +
 +
===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 01:38, 9 October 2022


EGFP-GGGSG-LBD

This composite part consists of an N-Terminal EGFP (BBa_K4414005) and a C-Terminal NR3C1 LBD (BBa_K4414000) domain. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.

Usage and Biology

We constructed a plasmid to link LBD with the fluorescent protein EGFP to verify the function of LBD. The EGFP on the N-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the C-Terminal is a ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a trans-activating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression[1].

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]



Fuctional test

Method

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding BBa_K4414031. 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 24 h or 48 h post glucocorticoids treatment. Finally, we observe the fluorescence intensity of cells.

Result

Fluorescence images are shown below, which indicates that glucocorticoids can bind to LBD and enter the nucleus. This provides a basic direction of thinking for our experiments.

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.