Composite

Part:BBa_K4414044

Designed by: Shiyin Jin   Group: iGEM22_NUDT_CHINA   (2022-09-29)
Revision as of 17:17, 11 October 2022 by TheaYang (Talk | contribs)


LBD-GSG-NES-GSG-TetR

This composite part consists of an N-terminal GR LBD(Part:BBa_K4414000) domain and a C-terminal tetR(Part:BBa_K4414009) domain fused with NES(Part:BBa_K4414003). 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 N 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) NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .

Figure1. Schematic figure of BBa_K4414044 and (Part:BBa_K4414041)



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]


Functional Test

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414044 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 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. (Shao, Qiu, & Xie, 2021)

Figure2.Schematic representation of the experimental process of validation for BBa_K4414044 and (Part:BBa_K4414041).

Result

Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (91.6 folds)(Figure 3).


Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414044.

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

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