Difference between revisions of "Part:BBa K4414044"
Line 44: Line 44:
  
 
</html>
 
</html>
−
 
+
Figure2.Schematic representation of the experimental process of validation for ([[BBa_K4414044]]) and ([[BBa_K4414041]]).
 
===Result===
 
===Result===
 
<html>
 
<html>
−
Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (5.63 folds)(Figure 2).
+
Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (91.6 folds)(Figure 3).
 
<figure class="figure">
 
<figure class="figure">
 
<img src="https://static.igem.wiki/teams/4414/wiki/44-3.png
 
<img src="https://static.igem.wiki/teams/4414/wiki/44-3.png
Line 57: Line 57:
  
  
−
Figure2. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by ([[BBa_K4414044]]).
+
Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by ([[BBa_K4414044]]).
  
 
==Reference==
 
==Reference==

Revision as of 06:22, 10 October 2022


LBD-GSG-NES-GSG-TetR

This composite part consists of an N-terminal NR3C1 LBD(BBa_K4414000) domain and a C-terminal tetR(BBa_K4414009) domain fused with NES(BBa_K4414003). It is designed to sense glucocorticoids 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. This part consists of a tetR DNA binding domain, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The NR3C1 LBD domain on the N terminal 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] NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .

Figure1. Schematic figure of (BBa_K4414044) and (BBa_K4414041)



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 Test

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both (BBa_K4414044) and TCE-SEAP(BBa_K4414041).

Method

Cells were treated with 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. [2]

Figure2.Schematic representation of the experimental process of validation for (BBa_K4414044) and (BBa_K4414041).

Result

Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (91.6 folds)(Figure 3).


Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by (BBa_K4414044).

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.