Composite-parts

In this year’s project, we want to build an engineered cell that would monitor the rising glucocorticoid level induced by chronic stress and responds accordingly with a visible output. We choose the stress hormone glucocorticoid as the indicator which will be detected when the pressure increases. In our registered and submitted parts, we provide a series of components using different combinations of components to sense glucocorticoids and activate the transcription of the reporter gene. Our favorite composite part LBD-GSG-NES-GSG-tetR （BBa_K4414044） constructed with an N-terminal GR_LBD (BBa_K4414000) and a C-terminal TetR (BBa_K4414009)  domain link with NES(BBa_K4414003).

As a glucocorticoid sensor, The GR_LBD (BBa_K4414000)  on the N terminal is the ligand-binding domain of the glucocorticoid receptor(GR), which 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) that can activate downstream gene expression (Weikum, Knuesel, Ortlund, & Yamamoto, 2017). The TetR (BBa_K4414009) on the C terminal consists of seven direct 19-bp Tet operator sequence (tetO) repeats that can bind to the TCE promoter (BBa_K4016011)   to activate downstream transcription. The NES (BBa_K4414003) is a nuclear export signal which can translocate protein from the nucleus into the cytosol. The above design achieves increased sensitivity to the response to glucocorticoid levels.

Figure 1. Schematic diagram of LBD-GSG-NES-GSG-tetR （BBa_K4414044）

To proof of the function of this composite part, we co-transfected human embryonic kidney (HEK-293T) cells with the plasmid encoding LBD-GSG-NES-GSG-tetR (BBa_K4414044) and the plasmid encoding TCE-SEAP. Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. The culture medium was collected at 48 h post glucocorticoids treatment. Detect the activity of SEAP in the medium according to a published protocol (Shao, Qiu, & Xie, 2021). 

Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (75.9 folds) and maintaining a nice dose dependency within the 0-100 nM glucocorticoid range.

Figure 2.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414044.