Difference between revisions of "Part:BBa K4414035"
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<partinfo>BBa_K4414035 short</partinfo> | <partinfo>BBa_K4414035 short</partinfo> | ||
| − | This composite part consists of an N-terminal tetR([[Part:BBa_K4414009]]) domain and a C-terminal | + | This composite part consists of an N-terminal tetR([[Part:BBa_K4414009]]) domain and a C-terminal GR LBD([[Part:BBa_K4414000]]) domain fused with a 3xGS linker([[Part:BBa_K4414063]]). 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 ([[Part:BBa_K4016011]]) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The | + | 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 GR 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(Weikum et al., 2017). |
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Figure1. Schematic figure of BBa_K4414035 and ([[Part:BBa_K4414041]]) | Figure1. Schematic figure of BBa_K4414035 and ([[Part:BBa_K4414041]]) | ||
| − | < | + | <!-- --> |
| + | ===Sequence and Features=== | ||
<partinfo>BBa_K4414035 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4414035 SequenceAndFeatures</partinfo> | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
| − | <partinfo> | + | <partinfo>BBa_K4414035 parameters</partinfo> |
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===Method=== | ===Method=== | ||
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| − | 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. | + | 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(Shao, Qiu, & Xie, 2021). |
<figure class="figure"> | <figure class="figure"> | ||
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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. 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 17:55, 11 October 2022
TetR-3xGSlinker-LBD
This composite part consists of an N-terminal tetR(Part:BBa_K4414009) domain and a C-terminal GR LBD(Part:BBa_K4414000) domain fused with a 3xGS linker(Part:BBa_K4414063). 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 GR 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(Weikum et al., 2017).
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_K4414035 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(Shao, Qiu, & Xie, 2021).
Result
Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (2.65 folds)(Figure 3).
Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414035.
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