Part:BBa_K4414029
EGFP-GGGSG-LBD
This composite part consists of an N-Terminal EGFP (BBa_K1123017) and a C-Terminal NR3C1 LBD (BBa_K4414000) domain. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.
Usage and Biology
We constructed a plasmid to link LBD with the fluorescent protein EGFP to verify the function of LBD. The EGFP on the N-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the C-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].
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_K4414029). Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. The fluorescence intensity of cells was observed 24 h after posting glucocorticoids treatment.
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.
Figure 2.The picture on the left is Bright-field cell diagram, the picture in the middle is fluorescence diagram, and the picture on the right is merge diagram.
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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