Difference between revisions of "Part:BBa K4414027"

Latest revision as of 17:51, 11 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 (Part: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 GR 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(Weikum et al., 2017).

Fugure1.Schematic figure of BBa_K4414027 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 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(Part: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(Shao, Qiu, & Xie, 2021).

Figure2.Schematic representation of the experimental process of validation for BBa_K4414027 and (Part: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, 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 9: / Line 9: @@
 ==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 ([[Part: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 GR<sub>LBD</sub> 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.(Weikum et al., 2017)
+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 ([[Part: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 GR 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(Weikum et al., 2017).
@@ Line 39: / Line 39: @@
 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([[Part: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.(Shao, Qiu, & Xie, 2021)
+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(Shao, Qiu, & Xie, 2021).
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