Difference between revisions of "Part:BBa K4414037"

Latest revision as of 17:55, 11 October 2022

TetR-GSG-NES-GSG-LBD

This composite part consists of a C-terminal tetR(Part:BBa_K4414009) domain and an GR LBD(Part:BBa_K4414000) domain fused with NES(Part: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 (Part:BBa_K4016011) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The GR 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(Weikum, Knuesel, Ortlund, & Yamamoto, 2017). NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .

Figure1. Figure1. Schematic figure of BBa_K4414037 and (Part: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 Validation

HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 and TCE-SEAP(Part: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 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol (Shao, Qiu, & Xie, 2021).

Figure 2:Cotransfected our plasmid with the TCE-SEAP into cells

Result

The test results are as follows:

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.

Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.7-2.0 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).

Reference

1. Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol, 18(3), 159-174. doi:10.1038/nrm.2016.152

2. Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol, 2312, 35-57. doi:10.1007/978-1-0716-1441-9_3

@@ Line 1: / Line 1: @@
 __NOTOC__
-<partinfo>K4414037 short</partinfo>
-This composite part consists of a C-terminal tetR(BBa_K4414009) domain and an NR3C1 LBD(BBa_K4414000) domain fused with NES(BBa_K4414003). It is designed to sense glucocorticoids and activates the transcription of the reporter gene.
+<partinfo>BBa_K4414037 short</partinfo>
+This composite part consists of a C-terminal tetR([[Part:BBa_K4414009]])  domain and an GR LBD([[Part:BBa_K4414000]]) domain fused with NES([[Part: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 .
+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 ([[Part:BBa_K4016011]]) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The GR 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(Weikum, Knuesel, Ortlund, & Yamamoto, 2017). NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .
 <html>
 <figure class="figure">
-<img src="https://static.igem.org/mediawiki/parts/1/17/T--NUDT_CHINA--Part_PixD-PixE_Schematic.png" class="figure-img img-fluid rounded"  height="250px">
+<img src="https://static.igem.wiki/teams/4414/wiki/037-1.png" class="figure-img img-fluid rounded"  height="350px">
 </figure>
 </html>
-Figure1. Schematic figure of PixE-PixD interaction under blue light stimulation
+Figure1. Figure1. Schematic figure of BBa_K4414037 and ([[Part:BBa_K4414041]])
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
+===Sequence and Features===
 <partinfo>BBa_K4414037 SequenceAndFeatures</partinfo>
@@ Line 35: / Line 37: @@
+==Functional Validation==
+HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 and TCE-SEAP([[Part: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 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol (Shao, Qiu, & Xie, 2021).
 <html>
 <figure class="figure">
-<img src="https://static.igem.org/mediawiki/parts/6/65/T--NUDT_CHINA--Part_Validation_SEAP_PixE-PixD.png
+<img src="https://static.igem.wiki/teams/4414/wiki/37-2.png" class="figure-img img-fluid rounded"  height="350px">
-" class="figure-img img-fluid rounded"  height="350px">
 </figure>
 </html>
-HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414044 and TCE-SEAP. Cells were treated with 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. SEAP activity was measured according to a published protocol. [2]
+Figure 2:Cotransfected our plasmid with the  TCE-SEAP into cells
 ===Result===
+The test results are as follows:
 <html>
 <figure class="figure">
-<img src="https://2021.igem.org/wiki/images/d/d6/T--NUDT_CHINA--Part_Result_00-01_.png
+<img src="https://static.igem.wiki/teams/4414/wiki/nudt2022-037-3.png" class="figure-img img-fluid rounded"  height="350px">
-" class="figure-img img-fluid rounded"  height="350px">
 </figure>
 </html>
-Figure 2 Result of SEAP test. The SEAP activity was calculated at 24h and 48h after transfection.
+Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.
-Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control. A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 1).
+Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.7-2.0 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).
+===Reference===
+. Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol, 18(3), 159-174. doi:10.1038/nrm.2016.152
+. Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol, 2312, 35-57. doi:10.1007/978-1-0716-1441-9_3
-===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.