iGEM 2022 — Team nudt-china

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notebook
[Monthly Progress & Milestones](#a)

![](https://static.igem.wiki/teams/4414/wiki/notebook.png)

NOTEBOOK

Monthly Progress & Milestones

***2021***

*December*

    ●Hold a biology contest to select team members for NUDT\_CHINA.

***2022***

*January*

    ●NUDT\_CHINA set-up. Team members split into 3 groups:Lab, Human Practice, Art&design.

*February*

    ●Conduct a questionnaire survey among more than 400 students.

    ●Brainstorm for 2022 team project.

    ●Learn the molecular biology and basic experimental technologies.

*March*

    ●Determine the direction of the 2022 iGEM project, consider to choose glococorticoids as our stress marker.

    ●Conduct preliminary Proof of Concept design and plasmid design.

*April*

    ●Collection and study of relevant literature on the application of our project.

    ●Hold online meeting with Fudan.

*May*

    ●Choose GRE as the module that respond to glucocorticoid stimulation.

*June*

    ●Offline meetings of the experimental group for literature reading, reporting the progress of the project and arranging the follow-up tasks.

*July*

    ●Hold online meeting with CSU\_CHINA.

    ●Online meetings of the experimental group for literature reading and plasmid design.

    ●Tried 3-copy GRE sequence and 6-copy GRE sequence in order to increase the response to glucocorticoid stimulation.

    ●Team uniform&project logo design.

*August*

    ●Conducting relevant experimental basic learning, including software design and skill training offline.

    ●Use EGFP to characterized the subcellular lolcalization of GR.

    ●Combine the advice and the feedback from literature to redesign the Proof of Concept to the right direction of improvemnet in increasing the response to glococorticoid stimulation.

    ●Construct the expression plasmid of EGFP and LBD fusion protein.

    ●Replace the lost DBD domain by using the DNA binding function of tetR.

    ●Promotion video production.

*September*

    ●Conduct offline Communication meetings with CSU\_CHINA and reach consensus on deep cooperation in collaboration, education and science communication.

    ●Establish partnership with CSU\_CHINA.

    ●Interview experts and receive optimization suggestions.

    ●Introduce Synthetic Biology to teenagers on the National Science Popularization Day in Changsha.

    ●Hold a disscussion with a cell biologist.

    ●Successfully complete the Proof of Concept and carry out the experimental design of Demonstration.

    ●Construct tdTomato and TYR expression plasmids regulated by tetO promoter.

    ●Complete the Demonstration experiment, feed the results back to Model group, and carry out the optimization design of experiment.

    ●Conduct optimized experiment with VP64 combined with NES and different length of linkers we designed.

    ●The optimized Demonstration experiment obtain better effect.

*October*

    ●Taking family photos.

    ●Conduct doctoral student interviews, research literature on application form and interview with clinical psychologist.

    ●Wiki construction.

    ●Presentation Video Production.

    ●Participation of Giant Jamboree.

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Engineering
[The First cycle: GR-based glucocorticoid-sensing circuit](#k)
[**The Second cycle: from GR to Tet****stress**](#T&L)
[**The Third cycle: Optimization of Tet****stress**](#R&R)

![](https://static.igem.wiki/teams/4414/wiki/engineering.png)

ENGINEERING

Overview

The engineering of a robust and sensitive stress hormone detection circuit is a crucial step in the development of the biomedical tattoo that monitors mental stress and offers an early warning to our users with a visible output. Through multiple design-test-learn cycles, we have managed to develop Tetstress, a novel synthetic transcriptional factor that effectively senses glucocorticoid, a well-established stress hormone whose subcutaneous level raises significantly upon chronic stress. In our [Results](https://2022.igem.wiki/nudt-china/results)  and [Proof of Concept](https://2022.igem.wiki/nudt-china/proof-of-concept)  pages, we have demonstrated the glucocorticoid-dose-dependent, visual signal produced by the Tetstress-based glucocorticoid-sensing circuit in engineered HEK-293T cells. In this Engineering Success page, we mainly discuss the design-test-learn cycles we went through during the development of the glucocorticoid-responsive transcriptional factor Tetstress (**Figure 1**).

![](https://static.igem.wiki/teams/4414/wiki/engineering-1.png)

***Figure 1. The Design-Test-Learn cycles we went through during the development of an effective Tet****stress.***

The First cycle: GR-based glucocorticoid-sensing circuit

**Design:** The most apparent approach to enable glucocorticoid sensing in mammalian cells is to rewire the endogenous glucocorticoid sensing pathways to ectopic gene expression.In humans, glucocorticoid receptor (GR) acts as a transcriptional activator in response to cortisol stimulation. Upon glucocorticoid stimulation, GRs translocate into the nucleus and bind specifically to the glucocorticoid response element (GRE), therefore initiating the expression of downstream genes. Utilizing GR as a glucocorticoid sensor, we designed chimeric promoters (PGRE3 and PGRE6) carrying either 3- or 6-copy GRE sequences upstream of a miniCMV promoter. Glucocorticoid-mediated activation of GR triggers the GR-PGRE binding and therefore activates downstream gene transcription (**Figure 2**).

![](https://static.igem.wiki/teams/4414/wiki/engineering-2.png)

***Figure 2.** **Human glucocorticoid receptor (GR)-based genetic circuits****.**Schematic representation of the GR-based glucocorticoid sensing circuit. Overexpressed GR binds to glucocorticoid, moves into the nucleus, and binds to a synthetic GR responsive promoter (PGRE), activating downstream gene expression.*

**Build and Test:** To evaluate the glucocorticoid-sensing performance of the GR-based glucocorticoid-sensing circuit, we co-transfected HEK-293T cells with the GR-expressing pNC046 plasmid and pNC047 (or pNC048) encoding PGRE3- or PGRE6-driven secreted alkaline phosphatase (SEAP)-expressing cassette. As shown in **Figure 3**, a mild upregulation of SEAP expression in cells treated with 100 nM glucocorticoid compared to the un-stimulated controls (1.92-fold for the PGRE3 group and 1.55-fold for the PGRE6 group) was observed. Cells treated with either 10 nM or 50 nM of glucocorticoids, however, showed no significant SEAP activation compared to the control cells.

![](https://static.igem.wiki/teams/4414/wiki/engineering-3.png)

***Figure 3.** **Glucocorticoid responsive transcriptional activation mediated by the Human glucocorticoid receptor (GR)-based genetic circuits.** PGRE-mediated SEAP production in glucocorticoid-stimulated HEK-293T cells co-transfected with pNC046 (PCMV-GR) and indicated reporter plasmids (pNC047 GRE3-Pmini-SEAP or pNC048 GRE6-Pmini-SEAP). SEAP production was measured 48 h post-DMEM or glucocorticoid stimulation; data shows mean±SD, n=3 independent experiments.*

To understand the cause of the low sensitivity, we linked the Enhanced Green Fluorescent Protein (EGFP) to GR, which allowed us to observe whether GRs were correctly imported into the nucleus upon glucocorticoid stimulation as previously reported. Surprisingly, we observed a robust nucleus-alike localization of the GR-EGFP protein in pNC049 (PCMV-GR-EGFP) transfected cells that were not even treated with glucocorticoids (**Figure 4**, left panel). Confocal images of HEK-293T cells co-transfected with actin-labeling pXQ100 (PCMV-lifeact-mcherry) and pNC049 (PCMV-GR-EGFP) also showed that GR-EGFP proteins were majorly located in an actin-free, nucleus-alike location (**Figure 4**, right panel) in both untreated control cells and glucocorticoid-treated cells.

![](https://static.igem.wiki/teams/4414/wiki/engineering-4.png)

***Figure 4.** **EGFP-labeled GR localization in HEK-293T cells w/wo glucocorticoid stimulation.** For wide-field microscopy, cells were transfected with pNC049 (PCMV-GR-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (PCMV-lifeact-mcherry) and GR labeling pWY049 (PCMV-GR-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid- administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.*

The Ligand Binding Domain (LBD) of human GR participants in activities including ligand binding, subcellular localization, and transcriptional activation. Therefore, we wondered whether there were some unknown components in HEK-293T that affected LBD, causing the incorrect location full-length GR had. Hence, we constructed a fusion protein comprising the GRLBD domain and EGFP (**Figure 5a**) to test our hypothesis. To our delight, both wide-field and confocal fluorescent imaging showed a broadly spread EGFP signal in the nucleus and cytoplasm of untreated, pNC051 (PCMV-GRLBD-EGFP) transfected HEK-293 cells. In contrast, a nucleus localization of GRLBD-EGFP can be observed in cells stimulated by glucocorticoid (**Figure 5b**). Taken together, these results suggested that GRLBD might still functional as a glucocorticoid-sensing element.

![](https://static.igem.wiki/teams/4414/wiki/engineering-5.png)

***Figure 5.** **Glucocorticoid-stimulated translocation of GR Ligand Binding Domain (LBD) in HEK-293T cells.** **(a)** Schematic representation of the GRLBD-EGFP construct. The LBD domain of human GR was fused to the N-Terminus of EGFP with a GGGSG linker. **(b)** EGFP-labeled GRLBD localization in HEK-293T cells w/wo glucocorticoid stimulation. For wide-field microscopy, cells were transfected with pNC051 (PCMV-GRLBD-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (PCMV-lifeact-mcherry) and GRLBD labeling pNC051 (PCMV-GRLBD-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.*

**Learn:** The imaging results of GR-EGFP surprisingly revealed that GRs might not be correctly located in HEK-293T cells, which is consistent to the SEAP assay showing a low sensitivity and poor transcriptional activation function. Luckily, the truncation of GR into GRLBD successfully rescued the glucocorticoid-responsive translocation of GRLBD-EGFP fusion protein. We thereby followed up this lead to design a new GRLBD-based glucocorticoid-sensing circuit.

**The Second cycle: from GR to Tet****stress**

**Design:** Building on the knowledge we obtained from the first engineering cycle, we then designed Tetstress, a novel synthetic transcriptional factor based on GRLBD. To reconstitute the DNA binding function of the lost GR DBD, we supplemented a DNA-binding tetR domain on either the C-terminus or the N-terminus of GRLBD. With this well-established DNA binding protein, we can also rewire the GR signaling to the well-established PTetO7 promoter. Furthermore, since the lost GR NTD domain carries a transcriptional activation function, we designed several variants adding the VP64 transcriptional activator as a replacement (**Figure 6**).

![](https://static.igem.wiki/teams/4414/wiki/engineering-6.png)

***Figure 6.** **Des****ign of** **tetR-GR****LBD****-based glucocorticoid responsive circuit.** tetR was fused to GRLBD on either its N-terminus or C-terminus. Optional VP64 domains were included to further improve transcriptional activation efficiency.*

**Build and Test:** To assess the function of the different Tetstress variants, we co-transfected the tetR reporter plasmid pXQ164 with a Tetstress variant (pNC054 Pcmv-tetR-GRLBD, pNC057 Pcmv-tetR-VP64-GRLBD, pNC056 Pcmv-GRLBD-tetR or pNC058 Pcmv-GRLBD-tetR-VP64) into HEK-293T cells. A major improvement of glucocorticoid response in both tetR-GRLBD and GRLBD-tetR configurations (5.21-fold for tetR-GRLBD and 22.50-fold for GRLBD-tetR compared to their respective non-treated controls) can be seen from the result. Meanwhile, the corresponding VP64-supplemented variants did not show significant enhancement in glucocorticoid response as we wished, which might be caused by the high background activation (**Figure 7a**). Further characterization of pNC054 (Pcmv-tetR-GRLBD) transfected cells illustrated that the tetR-GRLBD configuration resulted in a strong SEAP activation and a high basal expression, which significantly narrowed the dynamic range (approximately between 0-50 nM) of the circuit (**Figure 7b**). For the GRLBD-tetR configuration, HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP) and pNC056 (Pcmv-GRLBD-tetR) showed a much lower basal leakage compared to the tetR-GRLBD configuration, resulting in a significantly wider dynamic range (wider than 0-100 nM) and a better dose dependence (**Figure 7c**).

![](https://static.igem.wiki/teams/4414/wiki/engineering-7.png)

***Figure 7. Glucocorticoid responsive transcriptional activation mediated by the synthetic Tet****stress****transcriptional factor** **(a)** Glucocorticoid-activated SEAP production in HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP) and a tetR-GRLBD variant (pNC054 Pcmv-tetR-GRLBD, pNC057 Pcmv-tetR-VP64-GRLBD, pNC056 Pcmv-GRLBD-tetR or pNC058 Pcmv-GRLBD-tetR-VP64). SEAP production was measured 48 h post-DMEM or glucocorticoid stimulation; data shows mean value, n=2 independent experiments. **(b-c)** Glucocorticoid-dose-dependent SEAP production in HEK-293T cells mediated by the optimal tetR-GRLBD in **(a)**. Cells were co-transfected with pXQ164 (PTetO7-SEAP) and a tetR-GRLBD variant (pNC054 Pcmv-tetR-GRLBD for **c**, pNC056 Pcmv-GRLBD-tetR for (**c**). SEAP production was measured 48 h post-DMEM or glucocorticoid stimulation. Data shows mean±SD, n=3 independent experiments.*

**Learn:** Despite the significantly boosted glucocorticoid responsiveness of the Tetstress variants-based circuits, compared to the GR-based circuit, we still expect for higher sensitivity and signal intensity, especially for the GRLBD-tetR configuration. For this reason, we had a discussion with Mirta Viviani about our project, who is a Ph.D. student from Westlake University and experienced in engineering mammalian cell gene circuits [(see integrated HP page for more information)](https://2022.igem.wiki/nudt-china/human-practices). She suggested several potential approaches like optimizing PTetO7, changing linkers between tetR and GRLBD, and further manipulating the subcellular localization of the Tetstress transcriptional factor. Meanwhile, our modeling team performed a sensitivity screening among all the parameters that can be changed by circuit design. Their results showed that the circuit’s sensitivity largely depends on the efficiency of nuclear import of Tetstress upon glucocorticoid stimulation [(see Model page for more information)](https://2022.igem.wiki/nudt-china/model).

**The Third cycle: Optimization of Tet****stress**

**Design:** Building on the suggestions and findings we obtained in the previous engineering cycle, we then designed two new approaches that may improve Tetstress performance: 1) by optimizing the linkers between tetR and GRLBD, and 2) by integrating a nuclear export signal (NES) to manipulate the subcellular location ofTetstress. For the first approach, taking the tetR-GRLBD configuration as an example, we replaced the original 1xGS linker comprising a GGGSG sequence in the pNC054 (Pcmv-tetR-GRLBD) plasmid into a 3xGS ([GGGSG]3) or a 5xGS ([GGGSG]5) linker. Another configuration with no linker added between tetR and GRLBD was also generated (**Figure 8a**). For the second approach, a GSG-linker flanked NES was inserted between tetR and GRLBD in NES integration (**Figure 8b**).

![](https://static.igem.wiki/teams/4414/wiki/engineering-8.png)

***Figure 8.** **Linker-optimized tetR-GR****LBD****variant and NES-integrating tetR-GR****LBD****variant****(a)** Linker-optimized tetR-GRLBD variant (pNC064 Pcmv-tetR-[no linker]-GRLBD, pNC054 Pcmv-tetR-GRLBD, pNC065 Pcmv-tetR-[3xGS]-GRLBD, or pNC066 Pcmv-tetR-[5xGS]-GRLBD). **(b)** NES-integrating tetR-GRLBD variant (pNC054 Pcmv-tetR-GRLBD, pNC067 Pcmv-tetR-NES-GRLBD, pNC056 Pcmv-GRLBD-tetR or pNC069 Pcmv GRLBD-NES-tetR).*

**Build and Test:** While the removal of the linkers totally abolished the transcriptional activation activity of Tetstress, we were surprised to find that HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP), and one of these variants carrying GS linkers (pNC054 Pcmv-tetR-GRLBD, pNC065 Pcmv-tetR-[3xGS]-GRLBD, or pNC066 Pcmv-tetR-[5xGS]-GRLBD) showed a very similar glucocorticoid-stimulated SEAP expression level. We even observed an increasing basal SEAP production in configurations carrying longer GS linkers compared to the original 1xGS linker configuration (**Figure 9a**). Intriguingly, for the NES integrated Tetstress variants, while NES integration showed no significant improvement in the tetR-GRLBD configuration, a striking boost of glucocorticoid-stimulated SEAP production was observed in HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP) and pNC069 (PCMV-GRLBD-NES-tetR) compared to the cells transfected with the variant without NES (91.6x vs. 25x, compared to their untreated control). More importantly, the basal leakage SEAP expression remained similar between the two configurations(**Figure 9b**).

![](https://static.igem.wiki/teams/4414/wiki/engineering-9.png)

***Figure 9.** **Nuclear export signal (NES) allowed further improvement of Tet****stress****performance. (a)** Glucocorticoid-activated SEAP production in HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP) and a linker-optimized tetR-GRLBD variant (pNC064 Pcmv-tetR-[no linker]-GRLBD, pNC054 Pcmv-tetR-GRLBD, pNC065 Pcmv-tetR-[3xGS]-GRLBD, or pNC066 Pcmv-tetR-[5xGS]-GRLBD). **(b)** Glucocorticoid-activated SEAP production in HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP) and a NES-integrating tetR-GRLBD variant (pNC054 Pcmv-tetR-GRLBD, pNC067  Pcmv-tetR-NES-GRLBD, pNC056 Pcmv-GRLBD-tetR or pNC069 Pcmv GRLBD-NES-tetR). For panels **a** and **b**, SEAP production was measured 64 h post-DMEM or glucocorticoid stimulation. Data shows mean±SD, n=3 independent experiments.*

Shown above is the engineering process through which we built, tested, and optimized a synthetic glucocorticoid-responsive transcriptional factor from the scratch. Though in-depth characterization of this transcriptional factor is still on-going, we have already demonstrated its effectiveness and sensitivity. The further application of this transcriptional factor on stress-induced tattoo can be found in our [Results](https://2022.igem.wiki/nudt-china/results)  and [Proof of Concept](https://2022.igem.wiki/nudt-china/proof-of-concept)  pages.

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Experiment
[Molecular Part](#a)
[Cell Part](#b)

![](https://static.igem.wiki/teams/4414/wiki/experiment.png)

EXPERIMENTS

Molecular Part Protocol

LB-Medium-Preparation

Agarose Gel Electrophoresis

PCR-&-Agarose-Gel-Electrophoresis

Overlap-PCR-&-Agarose-Gel-Electrophoresis

Colony-PCR-&-Agarose-Gel-Electrophoresis

Gel-Purification

Double Enzyme DNA Digestion & Agarose Gel Electrophoresis

Infusion

Transformation

Plasmid conservation

Plasmid Extraction

Cell Part Protocol

Protocol-for-Cell-transfection-and-stimulation

Protocol-for-cell-culture-and-subculture

Protocol-for-seap-assay-in-vitro

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![](https://static.igem.wiki/teams/4414/wiki/results.jpg)

RESULTS

*GR-based genetic circuit enables weak glucocorticoid responsiveness in HEK-293T cells.*

In humans, glucocorticoids are sensed via glucocorticoid receptor (GR) encoded by the NR3C1 gene. In the absence of glucocorticoids, GRs are believed to form complexes with molecular chaperones like HSP90 in the cytosol. They translocate into the nucleus upon glucocorticoid stimulation and bind to Glucocorticoid responsive elements (GRE) in the promoter regions of the target genes (Weikum et al., 2017). Inspired by this endogenous signaling cascade, we designed a synthetic glucocorticoid-sensing circuit comprising GR and a chimeric promoter (PGRE) consisting of 3 or 6 tandem repeats of GREs upstream of a miniCMV promoter. Glucocorticoid-mediated activation of GR triggers the GR-PGRE binding and therefore activates downstream gene transcription (**Figure 1a**). To evaluate the glucocorticoid-sensing performance of this cascade, we co-transfected human embryonic kidney (HEK-293T) cells with the GR-expressing PNC046 plasmid and pNC047 (or pNC048) encoding PGRE-driven secreted alkaline phosphatase (SEAP)-expressing cassette. As expected, we observed a mild upregulation of SEAP expression in cells treated with 100 nM glucocorticoid compared to the un-stimulated controls (1.92-fold for the PGRE3 group and 1.55-fold for the PGRE6 group). However, cells treated with either 10 nM or 50 nM of glucocorticoids showed no significant SEAP activation compared to the control cells (**Figure 1b**), suggesting that these circuits might not be sensitive enough for subcutaneous glucocorticoid levels (approximately 1-10 nM).

Glucocorticoid-triggered nucleus entry of GR is a key step for GR-mediated transcriptional activation. We then went on to ask if the overexpressed GRs were correctly translocated into the nucleus upon glucocorticoid stimulation in our host cells. For this purpose, we constructed a GR-EGFP fusion protein to monitor the GR localization changes with the green fluorescent signal. Intriguingly, we observed a robust nucleus-like localization of the GR-EGFP protein in pNC049 (PCMV-GR-EGFP) transfected cells that were not even treated with glucocorticoids (**Figure 1c**, left panel). Confocal microscopy of HEK-293T cells co-transfected with actin-labeling pXQ100 (PCMV-lifeact-mcherry) and pNC049 (PCMV-GR-EGFP) also clearly showed that GR-EGFP proteins were majorly located in an actin-free, nucleus-alike location (**Figure 1c**, right panel) in both untreated control cells and glucocorticoid-treated cells. These findings surprisingly revealed that GRs might not be correctly located in HEK-293T cells, limiting its potential as a glucocorticoid sensor in this well-characterized host cell line.

![](https://static.igem.wiki/teams/4414/wiki/results-1.png)

***Figure 1. Human glucocorticoid receptor (GR)-based genetic circuits for glucocorticoid detection in HEK-293T cells.** **(a)** Schematic representation of the GR-based glucocorticoid sensing circuit. Overexpressed GR binds to glucocorticoid, moves into the nucleus, and binds to a synthetic GR responsive promoter (PGRE), activating downstream gene expression.**(b)** PGRE-mediated SEAP production in glucocorticoid-stimulated HEK-293T cells co-transfected with pNC046 (PCMV-GR) and indicated reporter plasmids (pNC047 GRE3-Pmini-SEAP or pNC048 GRE6-Pmini-SEAP). SEAP production was measured 48 h post-DMEM or glucocorticoid stimulation; data shows mean±SD, n=3 independent experiments. **(c)** EGFP-labeled GR localization in HEK-293T cells w/wo glucocorticoid stimulation. For wide-field microscopy, cells were transfected with pNC049  (PCMV-GR-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (PCMV-lifeact-mcherry) and GR labeling pWY049 (PCMV-GR-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid- administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.*

*Synthetic Tetstress transcriptional factor empowers robust glucocorticoid-inducible transcriptional activation.*

Human GRs share a similar domain structure consisting of an N-terminus domain (NTD), a zinc-finger DNA Binding Domain (DBD), a hinge region, and a C-terminus Ligand binding domain (LBD) (**Figure 2a**). The LBD domain is closely correlated to ligand binding, subcellular localization, and transcriptional activation. Since the full-length GR didn’t work well in HEK-293T cells, we then explored whether it was the LBD domain that got affected by the host cell. Hence, we constructed a fusion protein comprising the GRLBD domain and EGFP (**Figure 2a**). Interestingly, both wide-field and confocal fluorescent imaging of pNC051 (PCMV-GRLBD-EGFP) transfected cells showed that GRLBD-EGFP proteins were broadly spread all over the untreated control cells and were majorly nucleus-localized in glucocorticoid-stimulated cells (**Figure 2b**), suggesting the truncation of GR into GRLBD might be a possible approach to improve the sensitivity of a synthetic glucocorticoid-sensing circuit.

![](https://static.igem.wiki/teams/4414/wiki/results-2.png)

***Figure 2. Glucocorticoid-stimulated translocation of GR Ligand Binding Domain (LBD) in HEK-293T cells.** **(a)** Schematic representation of the GRLBD-EGFP construct. The LBD domain of human GR was fused to the N-Terminus of EGFP with a GGGSG linker. **(b)** EGFP-labeled GRLBD localization in HEK-293T cells w/wo glucocorticoid stimulation. For wide-field microscopy, cells were transfected with pNC051 (PCMV-GRLBD-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (PCMV-lifeact-mcherry) and GRLBD labeling pNC051 (PCMV-GRLBD-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.*

Building on this knowledge, we then designed Tetstress, a novel synthetic transcriptional factor, on the bases of GRLBD. Since we have abolished the DNA binding ability of GRLBD during the truncation, we then supplemented tetR, a well-characterized DNA binding protein, on the C-terminus or the N-terminus of the GRLBD to rewire the GR signaling to the well-established PtetO7 promoter. Also, since the NTD domain carries a transcriptional activation function, we designed several variants adding the VP64 transcriptional activator as a replacement (**Figure 3a**). To evaluate the function of these variants, we co-transfected HEK-293T cells with pXQ164 (PtetO7-SEAP) and a Tetstress variant (pNC054 PCMV-tetR-GRLBD, pNC057  PCMV-tetR-VP64-GRLBD, pNC056 PCMV-GRLBD-tetR or pNC058 PCMV-GRLBD-tetR-VP64). Results demonstrated a significantly improved glucocorticoid response in both tetR-GRLBD and GRLBD-tetR configurations (5.21-fold for tetR-GRLBD and 22.50-fold for GRLBD-tetR compared to their respective non-treated controls). At the same time, their corresponding VP64-supplemented variants showed poor glucocorticoid response mainly due to the high background activation (**Figure 3b**). Further characterization of pNC054 (PCMV-tetR-GRLBD) demonstrated that the tetR-GRLBD configuration resulted in a strong SEAP activation with a high basal expression, which significantly narrowed the dynamic range (approximately between 0-50 nM) of the circuit (**Figure 3c**). For the GRLBD-tetR configuration, HEK-293T cells co-transfected with pXQ164 (PtetO7-SEAP) and pNC056 (PCMV-GRLBD-tetR) showed a much lower basal leakage compared to the tetR-GRLBD configuration, resulting in a significantly wider dynamic range (wider than 0-100 nM) and a better dose dependence (**Figure 3d**).

![](https://static.igem.wiki/teams/4414/wiki/results-3.png)

***Figure 3. Glucocorticoid responsive transcriptional activation mediated by the synthetic Tet****stress****transcriptional factor** **(a)** Schematic representation of the TetR-GRLBD-based glucocorticoid responsive circuit. TetR was fused to GRLBD on either its N-terminus or C-terminus. Optional VP64 domains were included to further improve transcriptional activation efficiency. **(b)** Glucocorticoid-activated SEAP production in HEK-293T cells co-transfected with pXQ164 (PTetO7-SEAP) and a Tetstress variant (pNC054 Pcmv-TetR-GRLBD, pNC057 Pcmv-TetR-VP64-GRLBD, pNC056 Pcmv-GRLBD-TetR or pNC058 Pcmv-GRLBD-TetR-VP64). SEAP production was measured 48 h post-DMEM or glucocorticoid stimulation; data shows mean value, n=2 independent experiments. **(c-d)** Glucocorticoid-dose-dependent SEAP production in HEK-293T cells mediated by the optimal Tetstress in **(b)**. Cells were co-transfected with pXQ164 (PTetO7-SEAP) and a TetR-GRLBD variant (pNC054 Pcmv-TetR-GRLBD for **c**, pNC056 Pcmv-GRLBD-TetR for **d**). SEAP production was measured 24 or 48 h post-DMEM or glucocorticoid stimulation. Data shows mean±SD, n=3 independent experiments.*

*Tetstress-based HEKstress cells enable glucocorticoid sensing with fluorescent or colorimetric output*

With the GRLBD-tetR as the optimal configuration of the synthetic Tetstress transcriptional factor, we then went on to develop the HEKstress cells that generate fluorescent or colorimetric signals upon glucocorticoid stimulation, which can possibly serve as the basis of a biomedical tattoo. To enable fluorescent output, we constructed pNC059, a PtetO7-tdTomato reporter plasmid (**Figure 4a**). By co-transfecting HEK-293T cells with pNC060 (PCMV-GRLBD-tetR) and pNC056, we noticed a strong red fluorescent signal upon 100 nM glucocorticoid stimulation compared to the control cells treated by low concentration (1 nM) of glucocorticoid (**Figure 4b**). Since fluorescent signals were not visible to the naked eye, we changed the reporter gene into tyrosinase, the rate-limiting enzyme in synthesizing the black melanin pigment using Tyrosine as the substrate (**Figure 4c**). To fully demonstrate the potential of our glucocorticoid-sensing circuit, we generated a stable HEKstress cell line carrying both PtetO7-TYR and PCMV-GRLBD-tetRexpressing cassette with the sleeping beauty transpose system. Upon 96 hours of glucocorticoid stimulation, we observed dose-dependent enrichment of melanin pigment in HEKstress cells. Phase contrast imaging showed that melanin pigment gathered into spherical droplets within living cells, providing a possibly visible output while keeping the cells alive (**Figure 4d**). To further validate whether the output signals were actually visible, we trypsinized the cells and obtained cell pellets by centrifuge. As shown in **Figure 4e**, 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.

![](https://static.igem.wiki/teams/4414/wiki/results-4.png)

***Figure 4. Glucocorticoid responsive fluorescent or colorimetric output with HEK****stress** **cell. (a)** Schematic representation of HEKstress cell with a red fluorescent output signal.  **(b)** Glucocorticoid-activated fluorescent signal in HEK-293T cells co-transfected with pNC059 (PtetO7-tdTomato) and pNC060 (PCMV-GRLBD-tetR). Fluorescent images were taken 48 h post-glucocorticoid stimulation, scale bar: 100 µm. **(c)** Schematic representation of HEKstress cell with a black colorimetric output signal. In the lysosome, tyrosinase (TYR) generated by GRLBD-tetR activation transforms Tyrosine into melanin, thereby forming dark pigments. **(d)** Bright-field (ND) and Phase contrast (Ph) images of attached or trypsinized HEKstress cells under glucocorticoid stimulation. HEKstress cells stably express PtetO7-TYR and PCMV-GRLBD-tetR cassette.Images were taken 96 h post-glucocorticoid stimulation, scale bar: 100µm. **(e)** Cell pellets of the corresponding groups in panel **d**. For panels **b, d,** and **e**, data represent three independent experiments.*

*NES integration allows further optimization of Tetstressperformance*

With a solid proof-of-concept, we then went on to further characterize and optimize the performance of the Tetstress-based circuit. To figure out how to improve the sensitivity, we discussed our project with Mirta Viviani, a Ph.D. student from Westlake University. She is experienced in engineering mammalian cell gene circuits [(link to the human-practices page)](https://2022.igem.wiki/nudt-china/human-practices). She suggested several potential approaches like optimizing PtetO7, changing linkers between tetR and GRLBD, and further manipulating the subcellular localization of the Tetstress transcriptional factor. Meanwhile, our modeling team performed a sensitivity screening among all the parameters that can be changed by circuit design. Their results showed that the circuit’s sensitivity largely depends on the efficiency of nuclear import of Tetstress upon glucocorticoid stimulation [(link to the modeling page)](https://2022.igem.wiki/nudt-china/model#sen).

Building on these suggestions and findings, we then tested two potential approaches to improve Tetstress performance. On the one hand, we tried to manipulate the linker length between tetR and GRLBD. Taking the tetR-GRLBD configuration as an example, we replaced the original 1xGS linker comprising a GGGSG sequence in the pNC054 (PCMV-tetR-GRLBD) plasmid into a 3xGS ([GGGSG]3) or a 5xGS ([GGGSG]5) linker. Another configuration with no linker added between tetR and GRLBD was also generated (**Figure 5a**, upper panel). Unexpectedly, while the removal of the linker totally abolished the transcriptional activation function of Tetstress, a similar level of glucocorticoid-induced SEAP production was observed in HEK-293T cells co-transfected with pXQ164 (PtetO7-SEAP), and one of these variants carrying GS linkers (pNC054 PCMV-tetR-GRLBD, pNC065 PCMV-tetR-[3xGS]-GRLBD, or pNC066 PCMV-tetR-[5xGS]-GRLBD). We even observed an increasing basal SEAP production in configurations carrying longer GS linkers compared to the original 1xGS linker configuration (**Figure 5a**, lower panel).

On the other hand, we tried to include a nuclear export signal (NES) between tetR and GRLBD to manipulate the subcellular localization of the Tetstress (**Figure 5b**, upper panel). While NES integration showed no significant improvement in the tetR-GRLBD configuration, a striking boost of glucocorticoid-stimulated SEAP production was observed in HEK-293T cells co-transfected with pXQ164 (PtetO7-SEAP) and pNC069 (PCMV-GRLBD-NES-tetR) compared to the cells transfected with the variant without NES (91.6x vs. 25x, compared to their untreated control). At the same time, the basal leakage SEAP expression remained similar between the two configurations(**Figure 5b**, lower panel). To explore the nature of such a boost, we performed fluorescent imaging on cells transfected with the pNC069 (PCMV-GRLBD-NES-EGFP) plasmid to track the subcellular localization changes of the NES-integrated GRLBD. Interestingly, we observed a significantly improved cytosol localization and a reduced nuclear localization in the untreated cells (**Figure 5c**, upper row) compared to the GRLBD-EGFP configuration shown in **Figure 2**. We also observed a clear nucleus localization of GRLBD-NES-EGFP fusion protein in cells treated with 100 nM glucocorticoids (**Figure 5c**, lower row). These findings partly explained the boost of SEAP production in the NES-integrated Tetstress variant.

![](https://static.igem.wiki/teams/4414/wiki/results-5.png)

***Figure 5. Nuclear export signal (NES) allowed further improvement of Tet****stress****performance. (a)** Glucocorticoid-activated SEAP production in HEK-293T cells co-transfected with pXQ164 (PtetO7-SEAP) and a linker-optimized tetR-GRLBD variant (pNC064 PCMV-tetR-[no linker]-GRLBD, pNC054 PCMV-tetR-GRLBD, pNC065 PCMV-tetR-[3xGS]-GRLBD, or pNC066 PCMV-tetR-[5xGS]-GRLBD,as schematically demonstrated in the upper panel). **(b)** Glucocorticoid-activated SEAP production in HEK-293T cells co-transfected with pXQ164 (PtetO7-SEAP) and a NES-integrating tetR-GRLBD variant (pNC054 PCMV-tetR-GRLBD, pNC067  PCMV-tetR-NES-GRLBD, pNC056 PCMV-GRLBD-tetR or pNC069 PCMV GRLBD-NES-tetR,as schematically demonstrated in the upper panel). For panels **a** and **b**, SEAP production was measured 64 h post-DMEM or glucocorticoid stimulation. Data shows mean±SD, n=3 independent experiments. **(c)** EGFP-labeled NES-GRLBD localization in HEK-293T cells w/wo glucocorticoid stimulation. For wide-field microscopy, cells were transfected with pNC062 (PCMV-GRLBD-NES-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (PCMV-lifeact-mcherry) and GRLBD labeling pNC063 (PCMV-GRLBD-NES-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid- administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.*

Then, additional experiments were performed to profile the transcriptional activation kinetics and the glucocorticoid-dose dependence of NES-integrated Tetstress. As shown in **Figure 6a**, HEK-293T cells co-transfected with pXQ164 (PtetO7-SEAP) and pNC069 (PCMV-GRLBD-NES-tetR) produced a detectable amount of SEAP (0.69±0.15 U/L) at the 6th hour after 100 nM glucocorticoid stimulation, suggesting that short-term exposure to glucocorticoid might not be able to activate the Tetstress-mediated transcription, which nicely met our requirement for chronic stress detection. Also, pXQ164 and pNC069 co-transfected cells exposed to a gradient amount of glucocorticoid demonstrated that NES-integrated Tetstress would allow HEKstress cells to distinguish a 5 nM glucocorticoid difference within the 2-15 nM range (**Figure 6b**).

Due to the time limitation, we could not perform a thorough functional validation with fluorescent or colorimetric output using stable cell lines. Instead, we only demonstrated that NES-integrated Tetstress could effectively generate glucocorticoid-induced fluorescent or colorimetric responses even by transient transfection and under a lower glucocorticoid concentration (50 nM). As shown in **Figure 6c**, HEK-293T cells co-transfected with pNC069 (PCMV-GRLBD-NES-tetR) and pNC059 (PtetO7-tdTomato) showed a strong red fluorescent signal upon 50 nM glucocorticoid stimulation. In contrast, no fluorescent signal could be observed in the untreated cells. Similarly, 50 nM glucocorticoid stimulation could trigger a visible melanin production and accumulation in cells co-transfected with pNC069 (PCMV-GRLBD-NES-tetR) and pNC060 (PtetO7-TYR) (**Figure 6d**). Our future experiments will characterize the detection limits and dynamic range of the NES-integrated Tetstress.

![](https://static.igem.wiki/teams/4414/wiki/results-6.png)

***Figure 6. Characterization of NES-integrated HEK****stress****cells. (a)** SEAP production kinetics of HEKstress cells exposed to Glucocorticoid for a different amount of time. SEAP production was measured every 3 hours post-DMEM or glucocorticoid stimulation. Data shows mean±SD, n=3 independent experiments.**(b)** SEAP production in HEKstress cells exposed with increasing Glucocorticoid concentration. SEAP production was measured 48 h post-glucocorticoid stimulation. Data shows mean±SD, n=3 independent experiments. For panels **a** and **b**, HEKstress cells were generated by co-transfecting HEK-293T cells with pXQ164 (PtetO7-SEAP) and pNC069 (PCMV-GRLBD-NES-tetR).**(c)** Glucocorticoid-activated fluorescent signal in HEK-293T cells co-transfected with pNC059 (PtetO7-tdTomato) and pNC069 (PCMV-GRLBD-NES-tetR). **(d)** Columns 1 and 2: bright-field images of attached or trypsinized HEK-293T cells co-transfected with pNC060 (PtetO7-TYR) and pNC069 (PCMV-GRLBD-NES-tetR). Column 3: Pellet of corresponding cells shown in the left columns. For panels **c** and **d**, images were taken 48 h post-glucocorticoid stimulation, scale bar: 100 µm. Data were representative of 3 independent experiments.*

References

* Weikum, E.R., M.T. Knuesel, E.A. Ortlund, and K.R. Yamamoto. 2017. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol 18:159-174.