Proof of Concept

The ultimate goal of our project was to develop a synthetic biomedical tattoo with engineered cells that produce visible output in response to the rising glucocorticoid level induced by chronic stress. To achieve that goal, we engineered a novel synthetic transcriptional factor that rewires the glucocorticoid input to ectopic gene expression (see our Results and Engineering Success pages for detailed information). With this powerful tool, we then generated HEK_stress cells that efficiently sense glucocorticoids and produce fluorescent or colorimetric signals in a dose-dependent manner. Here as a proof of concept, we mainly demonstrate how different HEK_stress cells work upon glucocorticoid stimulation and show what the visual output looks like. Other results can be found on the Results page (see our Results page).

To enable effective glucocorticoid sensing in mammalian cells, we engineered a synthetic transcriptional factor Tet_stress which rewires glucocorticoid signal to a P_TetO7-driven ectopic gene expression (Figure 1a). Among the Tet_stress variants we developed, the GR_LBD-TetR configuration and the GR_LBD-NES-TetR demonstrated optimal glucocorticoid responsiveness (see our Engineering Success page for the details of the other variants). As shown in Figure 1b, HEK-293T cells co-transfected with pXQ164 (P_TetO7-SEAP) and pNC056 (P_CMV-GR_LBD-TetR) showed significant SEAP production in a glucocorticoid dose-dependent manner. Similarly, pXQ164 and pNC069 (P_CMV-GR_LBD-NES-TetR) co-transfected HEK-293T cells demonstrated a generally stronger SEAP activation compared to the GR_LBD-TetR configuration while maintaining a nice dose dependency within the 0-100 nM glucocorticoid range (Figure 1c). Results also showed that NES-integrated Tet_stress would allow HEK_stress cells to distinguish a 5 nM glucocorticoid difference within the 2-15 nM range (Figure 1c), which is sensitive enough for our proposed application.

Figure 1. Tet_stress-based gene circuits enable effective glucocorticoid-mediated ectopic gene expression. (a) Schematic representation of the glucocorticoid-responsive gene circuits with the optimal Tet_stress variants. (b-c) Glucocorticoid-dose-dependent SEAP production in HEK-293T cells mediated by the optimal Tet_stress variants. Cells were co-transfected with pXQ164 (P_TetO7-SEAP) and a Tet_stress variant (pNC056 P_CMV-GR_LBD-TetR for b and pNC069 P_CMV-GR_LBD-NES-TetR for c). SEAP production was measured 48 h after the indicated stimulation. Data shows mean±SD, n=3 independent experiments.

With an effective glucocorticoid-sensing circuit, we then went on to build HEK_stress cells with an easily detectable output signal. During our interview with our potential users, we noticed that some of them were worried about the potential privacy issues raised by a visible signal output (see our Human Practice and Proposed Implementation pages for more details). Hence, we developed a red fluorescent-producing HEK_stress to address their concerns by generating a P_TetO7-tdTomato reporter that couples the tdTomato expression to Tet_stress activation (Figure 2a). Fluorescent imaging of HEK-293T cells co-transfected with pNC056 (P_CMV-GR_LBD-TetR) and pNC059 (P_TetO7-tdTomato) showed a strong red fluorescent signal upon 100 nM glucocorticoid stimulation compared to the control cells treated by low concentration (1 nM) of glucocorticoid (Figure 2b). Similarly, with another optimal Tet_stress, HEK-293T cells co-transfected with pNC069 (P_CMV-GR_LBD-NES-TetR) and pNC059 (P_TetO7-tdTomato) also showed a strong red fluorescent signal upon 50 nM glucocorticoid stimulation (Figure 2c).

Figure 2. Glucocorticoid-responsive fluorescent production by HEK_stress cells. (a) Schematic representation of HEK_stress cell with a red fluorescent output signal. (b-c) Glucocorticoid-activated fluorescent signal mediated by the optimal Tet_stress variants. Cells were co-transfected with pNC059 (P_TetO7-tdTomato) and a Tet_stress variant (pNC056 P_CMV-GR_LBD-TetR for b and pNC069 P_CMV-GR_LBD-NES-TetR for c). Fluorescent images were taken 48 h post-glucocorticoid stimulation, scale bar: 100  µm.

Also, to provide another possible choice for our potential users, we also came up with another HEK_stress design with a visible signal. Herein, we put tyrosinase, the rate-limiting enzyme in synthesizing the black melanin pigment, under the control of the Tet_stress-based glucocorticoid sensing circuit (Figure 3a). To fully demonstrate the potential of our glucocorticoid-sensing circuit, we generated a stable HEK_stress cell line carrying both P_TetO7-TYR and P_CMV-GR_LBD-TetR expressing cassette with the sleeping beauty transpose system. Upon 96 hours of glucocorticoid stimulation, we observed dose-dependent enrichment of melanin pigment in HEK_stress cells (Figure 3b, left panel). To further validate whether the output signals were actually visible, we trypsinized the cells and obtained cell pellets by centrifuge. As shown in the left panel of Figure 3c, we observed a visible, dose-dependent color change on the cell pellet, firmly proving that our circuit may produce a tattoo-like function that effectively senses glucocorticoid. For the GR_LBD-NES-TetR configuration, which showed better performance compared to the GR_LBD-TetR configuration, we have not finished generating the stable cell line yet due to the limiting time of an iGEM session. But we have demonstrated that 50 nM glucocorticoid stimulation could trigger a visible melanin production and accumulation in cells co-transfected with pNC069 (P_CMV-GR_LBD-NES-TetR) and pNC060 (P_TetO7-TYR) (Figure 3b and 3c, right panel).

Figure 3. Glucocorticoid-responsive melanin production by HEK_stress cells. (a) Schematic representation of HEK_stress cell with a colorimetric output signal. (b-c) Glucocorticoid-activated colorimetric signal mediated by the optimal Tet_stress variants. For the GR_LBD-TetR configuration, stable cell lines expressing P_TetO7-TYR and P_CMV-GR_LBD-TetR cassette were used. For the GR_LBD-NES-TetR configuration, cells were co-transfected with pNC060 (P_TetO7-TYR) and pNC069 (P_CMV-GR_LBD-NES-TetR). Images were taken 48 h post-glucocorticoid stimulation, scale bar: 100  µm.

1. Comprehensive characterization of Tet_stress-based glucocorticoid circuit with stable cell lines (detection limit, dynamic range, sensitivity, signal intensity, ON-OFF dynamics, etc.).

2. Further evaluation of glucocorticoid-responsive melanin production in HEK_stressstable cell lines based on GR_LBD-NES-TetR configuration.

3. Cell encapsulation and characterization of encapsulated cells.

4. If everything works well, prepare an IACUC application to evaluate the function of HEK_stresscells in mouse depression or anxiety models.