Composite

Part:BBa_K4414038

Designed by: Rui Yang   Group: iGEM22_NUDT_CHINA   (2022-09-22)


LBD-GSG-NES-GSG-TetR-GGGSG-VP64

This composite part consists of a C-terminal GR LBD(Part:BBa_K4414000) ,a N-terminal VP64(Part:BBa_J176013) and a 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 C 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, Knuesel, Ortlund, & Yamamoto, 2017). NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol which helps to reduce nucleation rate in the absence of glucocorticoid stimulation to improve response efficiency. VP64 is a transcriptional activator consisting of four copies of VP16 (herpes simplex virus protein 16, amino acids 437-447*: DALDDFDLDML), linked with glycine-serine (GS). When fused to another protein structural domain that can bind near the gene promoter, VP64 functions as a powerful transcriptional activator.

Figure1: Schematic representation of the LBD-GSG-NES-GSG-tetR Part:BBa_K4414038 ’s function on binding to the TCE promoter and activating downstream transcription


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_K4414038 and TCE-SEAP(Part:BBa_K4414041).

Method

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. SEAP activity was measured according to a published protocol(Shao, Qiu, & Xie, 2021).

Figure2.Schematic representation of the experimental process of validation for BBa_K4414038 and (Part:BBa_K4414041).

Result

Results showed increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (4.72 folds). A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 2).

Figure 3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414038.

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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