Difference between revisions of "Part:BBa K4414028"
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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.[1] 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 (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. | + | 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.[1] 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 ([[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. |
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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-1*GSlinker-Tet R-VP64(BBa_K4414028) and TCE-SEAP([[BBa_K4414041]]). | + | To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both LBD-1*GSlinker-Tet R-VP64([[BBa_K4414028]]) and TCE-SEAP([[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 24h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2] | 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. [2] | ||
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| − | Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414028. | + | Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by ([[BBa_K4414028]]). |
===Reference=== | ===Reference=== | ||
Revision as of 16:11, 9 October 2022
LBD-GGGSG-tetR-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.[1] 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 (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.
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-1*GSlinker-Tet R-VP64(BBa_K4414028) and TCE-SEAP(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. [2]
Figure2.Schematic representation of the experimental process of validation for BBa_K4414028 and (BBa_K4414041).
Result
Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.78 folds).
Reference
[1].Weikum ER, Knuesel MT, Ortlund EA, Yamamoto KR. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol. 2017 Mar;18(3):159-174. doi: 10.1038/nrm.2016.152. Epub 2017 Jan 5. PMID: 28053348; PMCID: PMC6257982.
[2].Shao J, Qiu X, Xie M. Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol. 2021;2312:35-57. doi: 10.1007/978-1-0716-1441-9_3. PMID: 34228283.