Part:BBa_K4414043
LBD-GSG-NES-GSG-EGFP
This composite part consists of a C-Terminal EGFP (BBa_K1123017) and an N-Terminal NR3C1 LBD (BBa_K4414000) domain fused with a NES (BBa_K4414003 ). There is a GSG linker between every two genes. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.
Usage and Biology
The EGFP on the C-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the N-Terminal is a 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 trans-activating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression[1]. The NES is a nuclear export signal. To ensure that domains work properly, GSG linker is designed between every two genes.
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]
Fuctional test
Method
To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding BBa_K4414031. Cells were treated with 10, 50, or 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. Finally, we observe the fluorescence intensity of cells.
Result
Fluorescence images are shown below, which indicates that glucocorticoids can bind to LBD and enter the nucleus. This provides a basic direction of thinking for our experiments.
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.
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