Difference between revisions of "Part:BBa K4414036"
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<partinfo>K4414036 short</partinfo> | <partinfo>K4414036 short</partinfo> | ||
| − | This composite part consists of an N-terminal tetR([[BBa_K4414009]]) domain and a C-terminal NR3C1 LBD([[BBa_K4414000]]) domain fused with a 5xGS([[BBa_K4414064]]) linker. It is designed to sense glucocorticoids and activates the transcription of the reporter gene. | + | This composite part consists of an N-terminal tetR([[Part:BBa_K4414009]]) domain and a C-terminal NR3C1 LBD([[Part:BBa_K4414000]]) domain fused with a 5xGS([[Part:BBa_K4414064]]) linker. It is designed to sense glucocorticoids and activates the transcription of the reporter gene. |
==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. This part consists of a tetR DNA binding domain, which binds to the TCE promoter ([[BBa_K4016011]]) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The NR3C1 LBD domain on the C 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.[1] | + | 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 NR3C1 LBD domain on the C 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.[1] |
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| − | Figure1. Schematic figure of BBa_K4414036 and ([[BBa_K4414041]]) | + | Figure1. Schematic figure of BBa_K4414036 and ([[Part:BBa_K4414041]]) |
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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 | + | To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414036 and TCE-SEAP([[Part:BBa_K4414041]]). |
===Method=== | ===Method=== | ||
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| − | Figure2.Schematic representation of the experimental process of validation for | + | Figure2.Schematic representation of the experimental process of validation for BBa_K4414036 and ([[Part:BBa_K4414041]]). |
===Result=== | ===Result=== | ||
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| − | Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by | + | Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414036. |
==Reference== | ==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. | [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. | [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. | ||
Revision as of 07:07, 10 October 2022
tetR-5xGS linker-LBD
This composite part consists of an N-terminal tetR(Part:BBa_K4414009) domain and a C-terminal NR3C1 LBD(Part:BBa_K4414000) domain fused with a 5xGS(Part:BBa_K4414064) linker. 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 NR3C1 LBD domain on the C 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.[1]
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 BBa_K4414036 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 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2]
Result
Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (4.18 folds)(Figure 3).
Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414036.
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.