When the body is under chronic psychological stress, the hypothalamic–pituitary–adrenal (HPA) axis in the human body will be stimulated to release stress hormones, mainly glucocorticoids. The accumulation of glucocorticoids has been widely reported to be related to destructive outcomes like hair loss, anxiety, and depression. To achieve our ultimate goal of building a synthetic biological system that senses the stress hormones induced by chronic stress and responds accordingly with a visible output, we decided to generate an engineered cell that senses the rising glucocorticoid level induced by chronic stress produces chromoproteins. Once these cells are encapsulated and implanted subcutaneously, we can generate a tattoo-like pattern that changes color upon chronic stress conditions (Figure 1).
Figure 1. Schematic representation of the synthetic biomedical tattoo response to stress
As previously stated, the first part of our design focuses on sensing the pathologically high concentration of glucocorticoids representing chronic mental stress. Glucocorticoids are associated with several physiological processes, including the metabolism of nutrients and trace elements, mood, and cognitive functions, as well as immune responses, suggesting that there are canonical sensing pathways in human cell lines, with no exception in HEK293 cells. In humans, glucocorticoids are majorly sensed by the glucocorticoid receptor (GR), a nuclear receptor encoded by the NR3C1 gene. Without glucocorticoids, GRs are mainly in the cytoplasm as a multiprotein complex. After glucocorticoids enter the cell, they bind to the ligand binding domain of GR (GRLBD) and trigger a conformational change that leads to the nuclear import of GR. Then, the DNA binding domain of GR (GRDBD) binds to glucocorticoid-responsive elements (GREs) and activates the transcription of glucocorticoid-regulated genes.
Figure 2. Human glucocorticoid receptor (GR)-based genetic circuits
To fully utilize this pathway, we first obtained the DNA sequence that was recognized by GRDBD. We then built a chimeric promoter (PGRE) consisting of 3 or 6 tandem repeats of GREs upstream of a miniCMV promoter. We hypothesized that glucocorticoid-mediated activation of GR would trigger the GR-PGRE binding and therefore activates downstream gene transcription (Figure 2). However, results showed that this GR-based transcriptional circuit worked poorly in response to glucocorticoids, which is possibly due to the incorrect localization of GR in HEK-293T cells. Luckily, we did find that GRLBD can translocate as expected upon glucocorticoid stimulation.
To enable GRLBD-based transcriptional activation, we supplemented additional protein domains to reconstitute some missing functions caused by the truncation of full-length GR into GRLBD, thereby generating a novel transcriptional factor Tetstress. On the one hand, removing GRDBD leads to the loss of DNA binding function. To address this, we fused a TetR DNA binding protein to either the N- or the C- terminus of the GRLBD. This design would also significantly improve the orthogonality of the circuit since the original GR-regulated genes would not be affected by this synthetic transcriptional factor. On the other hand, GRNTD contains a transcriptional activation domain, whose removal might fail to activate downstream gene expression. Hence, we also designed some variants with transcriptional activator VP64 supplemented (Figure 3). Upon testing, we found that TetR-GRLBD and GRLBD-tetR configurations of Tetstress showed good signal intensity and glucocorticoid responsiveness (See Results page for more details).
Figure 3. Design of tetR-GRLBD-based glucocorticoid responsive circuit
This Tetstress sensor was then further optimized based on the suggestions we obtained during an interview performed by our HP members and the sensitivity analysis results of our modeling team . Herein, we tried to optimize the linkers between TetR and GRLBD. We also tried to manipulate the nuclear localization of the Tetstress to improve its performance (Figure 4).
Figure 4. Linker-optimized tetR-GRLBD variant and NES-integrating tetR-GRLBD variant
For a complete biomedical tattoo, it’s of great significance to be able to color the cells upon sensing the chronic stress markers. Thus, our next mission is to choose a non-toxic, bio-compatible pigmentary protein that is also easily observable and degradable. After literature research and numerous trials in HEK293 cells, tdtomato and tyrosine kinase stand out from a group of pigmentation genes and become our reporting genes.
Therefore, we replaced the SEAP gene in our reporter with the gene of tdtomato or tyrosine kinase, respectively, and co-transfect HEK293 cells with the improved sensing plasmid and reporter to managing the system of a pressure sensor (Figure 5).
Figure 5. Schematic representation of HEKstress cell