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 NR3C1 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 NR3C1 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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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 et al., 2021) |
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===Reference=== | ===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. Nature reviews. Molecular cell biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152 | |
| − | + | 2.Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods in molecular biology, 2312, 35-57 . | |
Revision as of 05:12, 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 NR3C1 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.
Sequecing
The plasmid was sequenced correct.
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 et al., 2021)
Figure2.Schematic representation of the experimental process of validation for BBa_K4414025 and (Part:BBa_K4414041).
Result
Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds).
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. Nature reviews. Molecular cell biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152 2.Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods in molecular biology, 2312, 35-57 .