Difference between revisions of "Part:BBa K4414044"

Revision as of 17:17, 11 October 2022

LBD-GSG-NES-GSG-TetR

This composite part consists of an N-terminal GR LBD(Part:BBa_K4414000) domain and a C-terminal tetR(Part:BBa_K4414009) 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 et al., 2017) NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .

Figure1. Schematic figure of BBa_K4414044 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 BBa_K4414044 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 48 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_K4414044 and (Part: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, 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 3: / Line 3: @@
 <partinfo>BBa_K4414044 short</partinfo>
-This composite part consists of an N-terminal GR<sub>LBD</sub>([[Part:BBa_K4414000]]) domain and a C-terminal tetR([[Part:BBa_K4414009]]) domain fused with NES([[Part:BBa_K4414003]]). It is designed to sense glucocorticoids and activates the transcription of the reporter gene.
+This composite part consists of an N-terminal GR LBD([[Part:BBa_K4414000]]) domain and a C-terminal tetR([[Part:BBa_K4414009]]) 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<sub>LBD</sub> 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 et al., 2017) 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 et al., 2017) NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .
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