Composite

Part:BBa_K4414031

Designed by: Bingshun Shi   Group: iGEM22_NUDT_CHINA   (2022-09-22)
Revision as of 17:09, 9 October 2022 by Bingshun (Talk | contribs)


EGFP-GSG-NES-GSG-LBD

This composite part consists of an N-Terminal EGFP (BBa_K1123017) and a C-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 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]. The NES is a nuclear export signal, which may help us reduce the background values. To ensure that domains work properly, GSG linker is designed to between every two genes.

Figure 1.Schematic figure of (BBa_K4414031)

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_K4414031). 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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