Basic-parts

In this year's project, we want to use synthetic biology to develop a stress detection system. We choose the stress hormone, glucocorticoid as the indicator which will be detected when the pressure increases. Glucocorticoid receptors (GRs) can function as a transcription factor that binds to glucocorticoid response elements (GREs) in the promoters of glucocorticoid-responsive genes to activate their transcription (Lu & Cidlowski, 2005). GR consists of an N-terminus binding domain (NTD), a DNA binding domain (DBD), and a ligand binding domain (LBD). As the major functional domain, the LBD domain is responsible for sensing glucocorticoid signals and translocating the protein into the nucleus (Monsalve et al., 2019). It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression (Weikum et al., 2017). Based on these functions of LBD, we use it as a sensor for the stress detection system.

Figure 1. Linear domain structure of glucocorticoid receptor (GR) (Weikum et al., 2017)

Our favourite part BBa_K4414000 is an important member of our functional parts，which can response to the stimulation of glucocorticoid, transfer from the cytoplasm to the nucleus and activate downstream gene transcription and translation.

To proof of the function of this basic part. We constructed a plasmid that linked LBD to the fluorescent protein EGFP. Cells are treated with 500 and 1000 nM glucocorticoids 6 h after transfection of plasmids into HEK-293T cells. Cells without glucocorticoid treatment were used as control. As the results shown in Figure2, glucocorticoid-treated cells fluorescence was mainly present in the nucleus compared to non-treated control, suggesting that glucocorticoids can bind to LBD and enter the nucleus.

Figure 2. GR_LBD-EGFP proteins were broadly spread all over the untreated control cells and were majorly nucleus-localized in glucocorticoid-stimulated cells.

For validation of LBD transcriptional activation capabilities, please refer to our registered composite element (BBa_K4414024), which consists of an N-terminal tetR (BBa_K4414009) domain and a C-Terminal LBD (BBa_K4414000) domain fused with a GGGSG linker. We co-transfected plasmids encoding both (BBa_K4414024) and TCE-SEAP to HEK-293T cells. 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. Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (2-5 folds) and a dose dependence was observed within 0-50 nM of glucocorticoid (Figure 3), suggesting that LBD can activate downstream gene transcription and translation.

Figure 3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by (BBa_K4414024).