Part:BBa_K4414037

TetR-GSG-NES-GSG-LBD

This composite part consists of a C-terminal tetR(Part:BBa_K4414009) domain and an NR3C1 LBD(Part:BBa_K4414000) 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 NR3C1 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, Knuesel, Ortlund, & Yamamoto, 2017) NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .

Figure1. Figure1. Schematic figure of BBa_K4414037 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 Validation

HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 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 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol (Shao, Qiu, & Xie, 2021).

Figure 2:Cotransfected our plasmid with the TCE-SEAP into cells

Result

The test results are as follows:

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.

Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control. A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).

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