Difference between revisions of "Part:BBa K4414027"

 
Line 1: Line 1:
 +
  
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4414027 short</partinfo>
 
<partinfo>BBa_K4414027 short</partinfo>
  
−
NO
+
This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.
  
−
<!-- Add more about the biology of this part here
+
 
−
===Usage and Biology===
+
==Usage and Biology==
 +
As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription.
 +
Tet R on the N terminus in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. The NR3C1 LBD domain on the C terminus is the 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 transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression.[1]
 +
 
 +
 
 +
<html>
 +
 
 +
<figure class="figure">
 +
<img src="https://static.igem.org/mediawiki/parts/5/5b/T--NUDT_CHINA--Part_SchematicFigure_15_.png" class="figure-img img-fluid rounded"  height="350px">
 +
 
 +
</figure>
 +
 
 +
</html>
 +
Fugure1.Schematic figure of BBa_K4414027 and BBa_K4414041.
 +
 
 +
 
 +
===Sequecing===
 +
The plasmid was sequenced correct.
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
===Sequence and Features===
−
<partinfo>BBa_K4414027 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4016015 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
−
<partinfo>BBa_K4414027 parameters</partinfo>
+
<partinfo>BBa_K4016015 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
 +
==Functional test==
 +
To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both Tet R-VP64-1*GS linker-LBD(BBa_K4414027) and TCE-SEAP(BBa_K4414041).
 +
===Method===
 +
Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol.[2]
 +
<html>
 +
 +
<figure class="figure">
 +
<img src="https://static.igem.org/mediawiki/parts/6/61/T--NUDT_CHINA--Part_Validation_CCK-8_72h.png
 +
" class="figure-img img-fluid rounded"  height="350px">
 +
 +
</figure>
 +
 +
</html>
 +
 +
Figure2.Schematic representation of the experimental process of validation for BBa_K4414027 and BBa_K4414041.
 +
 +
 +
===Result===
 +
Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.15 folds).
 +
 +
<html>
 +
 +
<figure class="figure">
 +
<img src="https://static.igem.org/mediawiki/parts/e/e5/T--NUDT_CHINA--Part_Result_15_.png
 +
" class="figure-img img-fluid rounded"  height="350px">
 +
 +
</figure>
 +
 +
</html>
 +
Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414027.
 +
===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.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 14:32, 8 October 2022


tetR-vp64-GGGSG-LBD

This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.


Usage and Biology

As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription. Tet R on the N terminus in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. The NR3C1 LBD domain on the C terminus is the 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 transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression.[1]


Fugure1.Schematic figure of BBa_K4414027 and BBa_K4414041.


Sequecing

The plasmid was sequenced correct.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 856
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 799
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 879



Functional test

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both Tet R-VP64-1*GS linker-LBD(BBa_K4414027) and TCE-SEAP(BBa_K4414041).

Method

Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol.[2]

Figure2.Schematic representation of the experimental process of validation for BBa_K4414027 and BBa_K4414041.


Result

Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.15 folds).

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414027.

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.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.