Difference between revisions of "Part:BBa K4414040"
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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. | ||
− | 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. The GR | + | 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. The GR LBD domain 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 (Weikum et al., 2017). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. |
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Revision as of 17:14, 11 October 2022
TetR-GGGSG-LBD-GGGSG-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. 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. The GR LBD domain 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 (Weikum et al., 2017). GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. Figure1.Schematic figure of BBa_K4414040 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 Tet R-LBD-1xGS linker-VP64(BBa_K4414040) 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 24h 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_K4414040 and (Part:BBa_K4414041).
Result
Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.92 folds).
Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414040.
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 .