Composite

Part:BBa_K4414043

Designed by: Bingshun Shi   Group: iGEM22_NUDT_CHINA   (2022-09-28)
Revision as of 17:10, 9 October 2022 by Bingshun (Talk | contribs)


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.

Figure 1.Schematic figure of (BBa_K4414043)

Sequecing

The plasmid was sequenced correct.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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_K4414043). 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 under the action of NES, 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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