Difference between revisions of "Part:BBa K4414025"
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==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. | 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. | ||
− | The GR LBD domain on the N 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 | + | The GR LBD domain on the N 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). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. Tet R 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. NLS (nuclear localization signal) helps the nucleophilic proteins better move into the nucleus. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. |
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==Functional test== | ==Functional test== | ||
− | To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64 BBa_K4414025 and TCE-SEAP([[Part:BBa_K4414041]]). | + | To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64 (BBa_K4414025) and TCE-SEAP([[Part:BBa_K4414041]]). |
===Method=== | ===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 | + | 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). |
<html> | <html> | ||
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Figure2.Schematic representation of the experimental process of validation | Figure2.Schematic representation of the experimental process of validation | ||
+ | |||
+ | ===Result=== | ||
+ | Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds). | ||
+ | <html> | ||
+ | |||
+ | <figure class="figure"> | ||
+ | <img src="https://static.igem.wiki/teams/4414/wiki/25-3.png" class="figure-img img-fluid rounded" height="350px"> | ||
+ | |||
+ | </figure> | ||
+ | |||
+ | </html> | ||
+ | Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414025. | ||
+ | |||
+ | |||
+ | |||
+ | ==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 |
Latest revision as of 18:21, 11 October 2022
LBD-GGGGGSG-tetR-GGGSG--NLS-vp64
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. The GR LBD domain on the N 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). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. Tet R 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. NLS (nuclear localization signal) helps the nucleophilic proteins better move into the nucleus. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers.
Figure 1. Schematic figure of BBa_K4414025 and (Part:BBa_K4414041).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64 (BBa_K4414025) 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
Result
Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds). Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414025.
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