iGEM 2024 — Team nudt-china

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## December 2023

Organizing campus promotions for iGEM 2024.

![](https://static.igem.wiki/teams/5267/notebook-yang/promotion.jpg)

## January 2024

Holding a biology contest to select team members for NUDT-CHINA 2024.

![](https://static.igem.wiki/teams/5267/notebook-yang/competition.png)

## Febuary 2024

Establishment of team NUDT-CHINA 2024. Team member chose their interesting groups according to their interests and advantages, including Wet Lab, Human Practice, Art&Design.

![](https://static.igem.wiki/teams/5267/notebook-yang/feb.jpg)

## March 2024

Conducting a literature review on industries associated with insomnia.

Conducting a questionnaire survey among over 2,000 participants.

Laboratory safety training.

![](https://static.igem.wiki/teams/5267/notebook-yang/survey-en.jpg)

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## April 2024

Brainstorming for 2024 team project.

Learning the molecular biology and basic experimental techniques.

Determining the initial direction of the 2024 iGEM project, considering to choose Melatonin as our fundamental molecule to design our platform. However, the core circuits to detect remained undecided.

![](https://static.igem.wiki/teams/5267/notebook-yang/brainstorm.jpg)
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## May 2024

Determining three circuits as the directions to design the platform for the detection of melatonin receptor agonists. Till now, our little knight had been ready for training to protect our sleep.

Conducting preliminary Proof of Concept design and plasmid design.

Start constructing MTNR1A plasmid with normal molecular strategy.

Start constructing NFAT plasmid with normal molecular strategy.

![](https://static.igem.wiki/teams/5267/may1.jpg)
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## June 2024

The NFAT plasmid was co-transfected with MTNR1A plasmid and MTNR1A-5xNFAT engineered cells were constructed by cis-transfection and assayed with melatonin.

Changing the multiplier, 1xNFAT, 6xNFAT and 7xNFAT corresponding engineered cells were constructed and assayed.

Development of the 1st generation engineered cell.

Detection of changes in intracellular calcium ion concentration in engineered cells using GCaMP plasmid transfected cells.

![](https://static.igem.wiki/teams/5267/notebook-yang/june1.jpg)
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## July 2024

Start constructing CRE plasmid with normal molecular strategy.

The CRE plasmid was co-transfected with MTNR1A plasmid and MTNR1A-4xCRE engineered cells were constructed by cis-transfection and assayed with melatonin.

Construction of MTNR1A-4xCRE-cAMP stabilized transient cell line

Changing the multiplier, 5xCRE and 6xCRE corresponding engineered cells were constructed and assayed.

![](https://static.igem.wiki/teams/5267/notebook-yang/july2.jpg)
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## August 2024

Screening of plant essential oils using MTNR1A-4xCRE-cAMP stable lines.

Molecular docking using MTNR1A-4xCRE-cAMP stable lines.

Production of the Promotion Video.

Creation of team giveaways and mascots.

Offline meeting of the wet lab group for further designing and engineering.

Start of production for the Science Popularization Picture Book.

![](https://static.igem.wiki/teams/5267/notebook-yang/august2.jpg)
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## September 2024

Start constructing MTNR1B plasmid with normal molecular strategy.

Start constructing ERK plasmid with normal molecular strategy.

The ERK plasmid was co-transfected with MTNR1B plasmid and MTNR1B-ERK engineered cells were constructed by cis-transfection and assayed with melatonin.

Team Roster Confirmed.

Team family photo.

WIKI Construction.

Interview with experts from the drug company. Informing us with detailed possible further application of our project.

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Dec (2023)

[January](#Jan1)

[Febuary](#Feb1)

[March](#Mar1)

[April](#Apr1)

[May](#May1)

[June](#June1)

[July](#July1)

[August](#Aug1)

[September](#Sep1)

[October](#Oct1)

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## Introduction

The design of a melatonin-responsive gene circuit and the construction of a designer-cell based drug screening assay are the key milestones of achieving engineering success in our project. Following the design-build-test-learn (DBTL) cycle (Figure 1) we have successfully constructed a drug screening plat from integrating in silico virtual screening and a mammalian cell-based assay. We have demonstrated that our screening pipeline helped identifying MO001, a constituent of EO016 essential oil, from 37 candidate essential oils we purchased from the market.. In this section, we describe in detail how this was achieved through multiple iterations of DBTL cycle.
Figure 1. Design-Build-Test-Learn Engineering Workflow (you can scroll horizontally to see more information)

![](https://static.igem.wiki/teams/5267/element/engineering-1758-1.png)

Figure 1.An Schematic Diagram of the Engineering

# Stage I: engineering of a melatonin-responsive gene circuit

## The First Cycle: MTNR1A switch V1.0 \_ Engineering of An MTNR1A receptor activation signal gene circuit in response to PLC/Ca2+pathway

### Design

As a G protein-coupled receptor, human melatonin receptor 1a (MTNR1A) has been identified to activate PKC/Ca2+ signaling1, which results in the elevation of intracellular Ca2+ levels. Hence, we designed a mammalian genetic circuit that utilizes the Nuclear Factors of Activated T cells (NFAT) to rewire the elevation of calcium signaling to transcriptional activation of the reporter gene. The elevated Ca2+ triggers the dephosphorylation of NFAT, which would then bind to the NFAT binding sites on the chimeric promoter and activate the downstream gene expression 2 (Fig. 2).
To simplify the quantification of reporter gene transcription activation, we used secreted Nanoluc luciferase (Nluc) as our reporter gene. By fusing Nanoluc coding gene with the DNA encoding secretion signaling peptide from IgK protein, we would be able to quantify the activation of target gene by measuring the Nanoluc activity in the culture medium.

![Figure 2](https://static.igem.wiki/teams/5267/element/1enfigure-2.png)

Figure 2. Schematic diagram of the gene circuit design that response to the MTNR1A-induced elevation of calcium level. The activation of MATNR1A triggers the elevation of intracellular Ca2+ level via the PLC/IP3 pathway; the elevated Ca2+ triggers the dephosphorylation of NFAT, which would then bind to the NFAT binding sites on the chimeric promoter and activate the downstream gene expression.

### Build & Test

To evaluate the functionality and sensitivity of our genetically engineered pathway, we co-transfected HEK293T cells with pNC099 (PCMV-MTNR1A), alongside either pNC102 (PNFAT\_1-IgK-Nluc), pNC100 (PNFAT\_5-IgK-Nluc), pNC104 (PNFAT\_6-IgK-Nluc), or pNC103 (PNFAT\_7-IgK-Nluc), which encode a PNFAT-driven Nluc-expressing cassette harboring different copy numbers of NFAT binding sites. Cells were then treated with 1 nM melatonin, a concentration that approximates physiological levels, at 6 hours post transfection. The nanoluc activity in the culture medium was measured after 48-hour to assess the melatonin-induced activation of reporter gene expression. Unfortunately, we observed no significant Nluc expression was observed in cells treated with melatonin compared to untreated controls (Fig. 3A). In contrast, robust Nluc expression could be detected in cells treated with Thapsigargin (Fig. 3B), a known inducer of intracellular calcium release via ER calcium depletion3. In addition, cells transfected with pNC107 (lacking NFAT binding sites) showed no significant response to Thapsigargin, confirming the specificity of the response. These findings suggest that MTNR1A may not effectively induce sufficient calcium signaling in HEK-293T cells to activate the NFAT-dependent calcium-responsive promoters.

![Figure 3](https://static.igem.wiki/teams/5267/shuiyin/engi-fig3.webp)

Figure 3. (A) The PNFAT promoters respond poorly to melatonin receptor activation. HEK293T cells were co-transfected with melatonin receptor plasmid pNC099(PCMV-MTNR1A) and either pNC102(PNFAT\_1-IgK-Nluc), pNC100(PNFAT\_5-IgK-Nluc), pNC104(PNFAT\_6-IgK-Nluc) or pNC103(PNFAT\_7-IgK-Nluc). Cells were treated with either DMSO or melatonin 6 hours post transcription. Data represents mean±SD of nanoluc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (B) The PNFAT promoters respond robustly to the Thapsigargin-induced elevation of intracellular calcium level. HEK293T cells were transfected with either pNC102(PNFAT\_1-IgK-Nluc), pNC100(PNFAT\_5-IgK-Nluc), pNC104(PNFAT\_6-IgK-Nluc) or pNC103(PNFAT\_7-IgK-Nluc). Cells were treated with either DMSO or Thapsigargin 6 hours post transcription. Data are mean±SD of nanoluc expression levels measured 48 h after thapsigargin stimulation (n = 3 independent experiments).

To investigate why the calcium-based circuit did not perform as expected, we consulted Dr. Yiming Dong, a synthetic biologist, and Chief Scientific Officer of Atantares. Drawing from her experience with calcium-sensing promoters in mammalian cells, Dr. Dong suggested that we examine the calcium dynamics triggered by melatonin receptors. She noted that, in her experience, most calcium-responsive promoters required a sustained elevation of intracellular calcium levels for proper activation. By utilizing GCaMP, a fluorescent protein that generate fluorescent signal in the existence of calcium ions4, we were able measured the intracellular calcium dynamics upon melatonin stimulation. We observed no significant change in GCaMP fluorescence in cells treated with melatonin for 48 hours compared to the untreated control cells (Fig. 4A and B), which is in consistent with data of the PNFAT-Nluc circuit. Interestingly, real-time live-cell calcium imaging revealed a significant spike in intracellular calcium levels immediately after melatonin administration. This calcium surge was short-lived, dissipating within 100 seconds of melatonin stimulation. In contrast, Thapsigargin induced a prolonged elevation of intracellular calcium levels (Fig. 4C).

![Figure 4](https://static.igem.wiki/teams/5267/shuiyin/engi-fig4.webp)

Figure 4. Melatonin-triggered alteration of intracellular Calcium dynamics in HEK-293T cells. (A and B) Long-term calcium response to melatonin stimulation in HEK-293T cells. HEK293T cells were co-transfected with the MTNR1A-expressing plasmid pNC099 (PCMV-MTNR1A) and the plasmid expressing the calcium indicator GCaMP6 (PCMV-GCaMP). Cells were treated with either DMSO or Melatonin 6 hours post transfection. For (A), Fluorescent images were taken 48 h after stimulation, scale bar: 100 µm. Data are representative images of 3 independent experiments. For (B), cells were subjected to fluorescence detection with a plate reader 48 h after stimulation. Data are mean±SD of relative fluorescence intensity (in RFU) measured 48 h after stimulation (n = 8 independent experiments). (C) Short-term calcium response to melatonin stimulation in HEK-293T cells. HEK293T cells were co-transfected with the MTNR1A-expressing plasmid pNC099 (PCMV-MTNR1A) and the plasmid expressing the calcium indicator GCaMP6 (PCMV-GCaMP) and treated with the indicated reagent at the 48 h post transfection. Cells were imaged through a fluorescent microscope every 5 seconds, and 20 cells were selected for fluorescent analysis in each group with ImageJ software. The cellular fluorescent levels of each group were normalized to the steady state level before chemical induction, Data shows Mean±SEM.

### Learn:

Our experimental results suggest that the design based on NFAT responding to the calcium signaling downstream of MTNR1A may not have met expectations because the calcium spike generate by melatonin cannot activate PNFAT efficiently. Since developing new calcium-inducible switches specifically for such calcium spike could be time-consuming, we decided to explore alternative signaling pathways.

## The Second Cycle: MTNR1A switch V2.0 \_Engineering of An MTNR1A receptor activation signal gene circuit in response to cAMP/PKA/CREB pathway

### Design

The MTNR1A receptor has the unique ability to couple to both Gs and Gi proteins, thereby enabling the bidirectional regulation of adenylyl cyclase and modulating cAMP signaling in a differential mannerr4,5.
The intricate balance of cAMP levels, in turn, influences the activation state of CREB (cAMP-response element-binding protein), which acts as a pivotal transcription factor in various cellular processes, including gene expression related to metabolism, cell survival, and neuronal plasticity.

We have engineered a novel genetic circuit utilizing the cAMP-responsive element (CRE) as the signaling responsive element to drive the expression of the downstream reporter gene NanoLuc.
This genetic circuit is capable of reporting the upregulation of cAMP signals within cells, based on our literature-derived confirmation that upon melatonin stimulation, cAMP levels in HEK293 cells are upregulated rather than downregulated following the activation of MTNR1A5.(Figure 5)

![Figure 5](https://static.igem.wiki/teams/5267/element/1enfigure-5.png)

Figure 5. Schematic diagram of the gene circuit design that responds to the MTNR1A-induced alteration of cAMP level. The activation of MATNR1A triggers the elevation of intracellular cAMP level, which triggers the activation of CREB, which would then bind to the CRE sites on the chimeric promoter and activate the downstream gene expression.

### Build & Test

To assess the functionality and sensitivity of our genetic circuit, we engineered a series of genetic constructs incorporating varying copy numbers of the cAMP-responsive element (CRE) as the reporter component and assessed the expression efficiency of NanoLuc (Nluc) under the stimulation of 1 nM melatonin
Our results demonstrated that the CRE element-based design responded effectively to the increased intracellular cAMP levels, with the tetrameric CRE element showing the most robust response (Figure 6).
It is gratifying that the group without melatonin stimulation exhibited a relatively low basal level of NanoLuc expression.
In contrast, upon stimulation with 1 nM melatonin, the cells stably and robustly expressed the nanoluciferase protein.
This confirms that the genetic circuit we designed is responsive to the activating signal of melatonin on its receptor and can stably report it for our detection

![Figure 6](https://static.igem.wiki/teams/5267/shuiyin/engi-fig6.webp)

Figure 6. Co-expression of MTNR1A and PCRE-promoter variants enables robust transcriptional activation upon melatonin stimulation. HEK293T cells were co-transfected with pNC099(PCMV-MTNR1A) and either pNC101(PCRE\_4-IgK-Nluc), pNC105(PCRE\_5-IgK-Nluc), or pNC106(PCRE\_6-IgK-Nluc). Cells were treated with DMSO or melatonin at 6 hours post transfection; data are mean±SD of nanoluc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments).

To further characterize the melatonin-activating circuit, we generated a stable HEKMT cell line carrying both PCRE4-Nluc and PCMV-MTNR1A ¬expressing cassette with the sleeping beauty transpose system. As shown in Fig. 7A, HEKMT cells exposed to a gradient amount of melatonin could produce detectable amount of Nluc at around the 12th hour in a dose-dependent manner, with a EC50 of 0.4228 nM at the 24 h post melatonin stimulus (Fig. 7B). Additionally, we also showed that HEKMT cells are also capable of responding dose-dependently to Ramelteon, a commercially available melatonin receptor agonist (Fig. 7 C). Together, these findings demonstrated the feasibility of the HEKMT cells as the basis of a drug-screening platform.

### Learn

To this end, we have demonstrated the success engineering of a melatonin-responsive genetic circuit in mammalian cells. However, it came to our knowledge that the circuit itself is not sufficient for us to build a cell-based drug screening platform.
During our interview with Dr. Yiming Dong, she mentioned that the endogenous signaling pathways we chose (eg. Ca2+ and cAMP/PKA pathways) are also tightly coupled with a set of endogenous signaling pathways, which might also be affected by the compound we add into the cells. Therefore, we should also design a control assay to identify these potential false-positive hits.

# Stage II: Engineering of a drug screening platform to platform to discover MTNR1A agonist

## The Third Cycle: MTNR1A agonist Screening platform V1.0\_Engineering of A Drug screening platform based on HEKCTR and HEKMT

### Design

Building on the melatonin-responsive circuits, we then went on to design a cell-based drug screening platform to discover MTNR1A agonist. Learning from the interview with Dr. Dong, we design a platform consists of two stable cell lines, HEKMT and HEKCTR The HEKMT cells express both MTNR1A and PCRE-Nluc cassette, while the HEKCTR cells were incorporated with PCRE-Nluc cassette only. During the screening process, we would treat both cells simultaneously with identical compound library. The ones that activate Nluc expression in HEKMT instead of HEKCTR cells will be considered as potential hits.

### Build & Test

To generate the 1.0 version of MTNR1A agonist screening platform, we first generated the HEKMT and HEKCTR cell lines with the sleeping beauty transposon system. Then, we decided to validate the feasibility of our platform by running a minibatch of screening. While we were building the HEKCTR cells, we interviewed the front-line psychiatrists and engaged the potential user of the drug we might discover through our screening platform to gain some hints about what we can start testing our screening platform on. Interestingly, the term “aromatherapy” pops up as its been widely used around the globe for thousands of years to help falling asleep, and the general public feels more comfortable using these natural product as a daily supplement (See Human Practices page for more information). Hence, we purchased 37 plant essential oils online and hope to see if we can get lucky finding some new agonists for MTNR1A.

![Figure 7](https://static.igem.wiki/teams/5267/shuiyin/engi-fig7.webp)

Figure 7. The characterization of a cAMP/CREB-based melatonin-responsive gene circuit in HEK-293T cells. (A) Finetuning of PCRE4-Nluc allows the tuning of system behavior. HEK293T cells were co-transfected with pNC099(PCMV-MTNR1A) and pNC101(PCRE\_4-IgK-Nluc) under the indicated plasmid ratio. Cells were treated with DMSO or melatonin at 6 hours post transfection. Data are mean±SD of nanoluc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (B) Step-response dynamics of HEKMT cells under melatonin treatment. HEKMT cell line carrying both PCRE4-Nluc and PCMV-MTNR1A expressing cassette was stimulated with the indicated amount of melatonin 16 hours post seeding. Nanoluc expression levels were measured at different time points after melatonin stimulation (Data are mean±SD, n = 3 independent experiments). (C) Dose-dependence curve of HEKMT cells under melatonin treatment. HEKMT cell line was stimulated with the indicated amount of melatonin 16 hours post seeding. Nanoluc expression levels were measured at 24 h after melatonin stimulation (Data are mean±SD, n = 3 independent experiments, EC50=0.4228 nM). (D) Ramelteon response of the HEKMT cells. HEKMT cells were treated with ramelteon 16 h post seeding. Nanoluc expression levels were measured at 24 h after ramelteon stimulation (Data are mean±SD, n = 3 independent experiments).

Intriguingly, while most of the plant essential oils showed no significant alteration in the Nluc production of either the HEKMT cells or the HEKCTR cells, we surprisingly found that the HEKMT cells treated with EO016 oil showed a robust elevation in Nluc production compared to the DMSO-treated control (~5.25-fold). In comparison, Nluc production in HEKCTR cells showed no significant alteration, suggesting that EO016 oil activates Nluc production in a MTNR1A-dependent manner (Fig. 8A). Similar results were also observed on EO017 Oil, with a milder induction rate of Nluc production in HEKMT cells (~2.32-fold, Fig. 8A). Further analysis revealed that both EO016 oil and EO017 Oil could robustly induce MTNR1A-dependent Nluc production, but no clear dose-dependence could be observed on EO017 Oil (Fig. 8B and 8C). These findings not only showed the feasibility of our designer-cell-based platform for rapid, robust, and resource-efficient high-throughput drug screening but also identified two essential oil hits for agonist discovery.
(Figure 8)

![Figure 8](https://static.igem.wiki/teams/5267/engi-fig8-bianhao.webp)

Figure 8. Designer cell-based high throughput screen of plant essential oils to identify MTNR1A agonism. (A) High-throughput analysis. HEKCTR refers to stable HEK293T cells stabling expressing PCRE\_4-Nluc, HEKMT refers to the HEK293T cells shown in Fig. 3D-3F. Cells were treated with 1×104 v/v of either DMSO or indicated essential oil 16 h post seeding. Cells treated with 1 nM melatonin were used as positive control. Nanoluc expression levels were measured at 24 h after exposure to essential oils (Data are mean±SD, n = 3 independent experiments). (B, C) Dose-dependent validation of the most active essential oil hits. (B) HEKCTR and (C) HEKMT were treated with different essential oil dilutions (v/v) 16 h post seeding. Nanoluc expression levels were measured at 24 h after exposure to essential oils (Data are mean±SD, n = 3 independent experiments).

### Learn

Most essential oils are a mixture of multiple natural product molecules contributing differently to the overall therapeutic function. However, screening over all constituents in the essential oil hits to identify the functioning molecule can sometimes be difficult due to the availability of the constituents and the time- or financial-constraints. Similar concerns were also raised by Dr. Li, the instructors of HZAU-CHINA, during our presentation in the Central China iGEMer’s Meetup in Wuhan, China. Dr.Li suggested us to screen over the essential oil constituents in silico before moving towards the cellular experiments (refer to the Human Practice Page for more information).
Almost concurrently, through interviews with experts and scholars in various fields, we have noticed that we can pay more attention to MTNR1B, as selective melatonin receptor agonists have a broader and safer application prospect in clinical therapy. Therefore, we plan to introduce both MTNR1A and MTNR1B in the virtual screening.

# The Forth Cycle: Engineering of A drug virtual screening platform for selective MTNR1A receptor agonists by molecular docking

### Design

To achieve a more robust and efficient identification of the putative active constituents in the essential oils that account for the activation of melatonin receptors, we generated an AutoDock Vina6-based pipeline to predict the binding affinity of the essential oil constituents against the known structure of melatonin receptor 1A (PDB ID 6ME2). Based on the input from our human practices, we also included the docking process with melatonin receptor 1B (PDB ID 7VH0) to identify the possible selectivity between MTNR1A and MTNR1B (Fig. 9). Following the previous iteration, we then validated the functionality of the improved screening platform by identifying the functional constituents from the essential oil hits we generated in our last in vitro screening.

![Figure 9](https://static.igem.wiki/teams/5267/shuiyin/engi-fig9.webp)

Figure 9. (A) Docking box for small molecules with the MTNR1A receptor protein. (B) Docking box for small molecules with the MTNR1B receptor protein.

### Build & Test

To validate the molecule docking parameters, we first preformed docking utilizing some known binders of MTNR1A and MTNR1B. The result suggest that CTL 01-05-B-A05 is a highly potent binder to both MTNR1A and MTNR1B (Fig. 10A, upper panel, also see the Modeling page for more information), outperforming other ligands in binding affinity. Notably, It displays a clear preference for MTNR1A over MTNR1B. In contrast, melatonin was found to be a relatively weak binder towards MTNR1A/MTNR1B receptors, consistent with its role in promoting sleep and being naturally degraded during the sleep cycle. These findings validate the feasibility of the docking parameters employed.
Then, we performed in silico screening of all constituents of EO016 and EO017 essential oils by molecular docking. Interestingly, the major constituents of EO016 oil were found to bind to both MTNR1A/ MTNR1B receptors. Only MO001 exhibited a preference towards MTNR1A (Fig. 10A). Structural analysis revealed that the hydroxy group of MO001 may form a hydrogen bond with the main chain oxgen of Ala104 in MTNR1A (Fig. 10B).
More importantly, in vitro assay showed a significantly increased Nluc production in HEKMT cells treated with MO001, while no Nluc induction was observed in HEKCTR cells (Fig. 10C). Together, these results demonstrated the feasibility of our drug-screening pipeline that incorporates both designer cell-based in vitro screening and molecular docking-based in silico screening, and also suggested that MO001 might serve as a promising selective agonist for MTNR1A.

![Figure 10](https://static.igem.wiki/teams/5267/engi-fig10-bianhao.webp)

Figure 10. In silico and in vitro screening of MTNR1A selective constituents from essential oils. (A) Heatmap of binding affinities of various melatonin receptor agonists and natural small molecules from EO016 and EO017 towards MTNR1A and MTNR1B. The main constituents’ ratios were presented within parentheses. (B) Structural visualization of MO001 binds with MTNR1A (left panel). MTNR1A protein is shown as cartoon, displayed as rainbow colors by different helix bundle. MO001 molecule is shown as magenta sticks. 2D-interaction of MO001 with MTNR1A (right panel). Potential hydrogen bond is shown between MO001 and Ala104. (C) Quantification of MTNR1A agonism by MO001 using designer cells. HEKCTR (left) and HEKMT (right) were treated with the indicated concentration of MO001 16 h post seeding. Nanoluc expression levels were measured at 24 h after MO001 induction (Data are mean±SD, n = 3 independent experiments).

### Learn

Although we have demonstrated the feasibility of our screening platform, we have also noticed that it is necessary to further incorporate the MTNR1B receptor-based cell assay so we can further validate the selectivity of drug candidates experimentally.
Unfortunately, due to time constraints, we could not complete the construction of the new circuit before the WIKI FREEZE deadline. We have decided to keep going on designing the MTNR1B-responsive circuit after iGEM.

## References

1

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Okamoto, H. H., Cecon, E., Nureki, O., Rivara, S. & Jockers, R. Melatonin receptor structure and signaling. J Pineal Res 76, e12952 (2024). https://doi.org/10.1111/jpi.12952

Muller, M. R. & Rao, A. NFAT, immunity and cancer: a transcription factor comes of age. Nat Rev Immunol 10, 645-656 (2010). https://doi.org/10.1038/nri2818

Jaskulska, A., Janecka, A. E. & Gach
-Janczak, K. Thapsigargin-From Traditional Medicine to Anticancer Drug. Int J Mol Sci 22 (2020). https://doi.org/10.3390/ijms22010004

Zhang, Y. et al. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature 615, 884-891 (2023). https://doi.org/10.1038/s41586-023-05828-9

Conway, S. et al. Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein-coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels. FEBS Lett 407, 121-126 (1997). https://doi.org/10.1016/s0014-5793(97)00315-3

Tse, L. H., Cheung, S. T., Lee, S. & Wong, Y. H. Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G(s) Protein to the Melatonin MT(1) Receptor. Int J Mol Sci 25 (2024). https://doi.org/10.3390/ijms25052919

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Model

[Introduction](#block1)

[The First Cycle](#block2)

[The Second Cycle](#block3)

[The Third Cycle](#block4)

[The Forth Cycle](#block5)

[References](#ref-block)

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## Molecular Experiments

**Agarose Gel Electrophoresis**
**Material**
TAE buffer
Agarose Powder
Ethidium Bromide (EB)
Comb and Bed
**Step (take 30ml gel for instance)**
① Weigh 0.3g agarose powder and 30μl 1%TAE buffer, then add to a flask;
② Heat up until the solution is completely dissolved. If it boils, move away from the heat until it cools down and put it back on the heat until it absolutely dissolved.
③ While heating, prepare the bed where the gel polymerizes. Make sure that it is well balanced and tight, and that the comb is well placed.
④ When the solution cools down to approximately 55 ℃ , add 1µl EB to the homogeneous
solution and mix well.
⑤ Pour the solution into the bed and clear all the bubbles in it with a tip.
⑥ When the gel solidification solidifies, carefully pull out the “comb”;
⑦ Place the gel in the electrophoresis chamber, then add enough TAE buffer till there is about 2-3 mm of buffer over the gel.
⑧ Mix the samples with loading buffer in a 10:1 ratio, then put the samples into the wells, as
well as marker into the first well.
⑨ Run the gel at 120V for about 30 minutes, then check the result under the Blue Light Gel
Imager.
**Note**
① For different size of gels, we have 25 ml,50 ml and 100 ml agarose gel.
② We often use 0.7%,1% and 1.5% agarose gel for different samples.

**Double Enzyme DNA Digestion& Agarose Gel Electrophoresis**
**Material**
Plasmid DNA
10× FastDigest or 10× FastDigest Green Buffer
Double Distillation Water (ddH2O)
FastDigest enzyme
Alkaline phosphatase
**Steps**
① Add 1μg plasmid DNA, 1μl FastDigest enzyme, 2μl 10× FastDigest or 10× FastDigest
Green Buffer and ddH2O to 20μl into an PCR tube.
② Put the tube into the oven at 37℃ for 1h to 1.5h.
③ Add 1μl Alkaline phosphatase.
④ Put the tube into the oven at 37℃ for 0.5h.
⑤ Stop the reaction by placing the tube at -4°C for 5 to 10 minutes.
⑥ Agarose Gel Electrophoresis.
⑦ Store the tube into the refrigerator at -20℃.

**Gel Purification**
**Material**
TIANGEN Universal DNA Purification Kit

Gel with DNA sample

Double Distillation Water (ddH2O)

Centrifuge

Shaker

Water Bath

**Steps**
① Cut the gel that DNA sample lies with a knife.

② Put the gel into an EP tube, add PC solution, which has the same volume as the gel.
Shake the tube at 50℃ for 10min till the gel is completely dissolved and the solution
color is yellow.

③ Insert the adsorption column into a collection tube, add 500μl BL equilibrium solution,
centrifuge at 12,000G for 1min, discard the filtrate and reuse the collection tube.

④ Add the solution to the adsorption column, centrifuge at 12,000G for 1min, discard the
filtrate and reuse the collection tube.

⑤ Add 600μl PW rinse solution, let stand for 5min, centrifuge at 12,000G for 1min,
discard the filtrate and reuse the collection tube.

⑥ Repeat step⑤.

⑦ Put the adsorption column into a clean EP tube, centrifuge at 12,000G for 2min, discard
the filtrate. Open the cover of adsorption column and dry it out in the air for 5min.

⑧ Add 100μl ddH2O (preheated to 70 ℃ in advance), let stand for 2min, and centrifuge
at 12,000G for 2 min.

⑨ Add filtrate to the adsorption column again, let stand for 2min, and repeat step⑧.

⑩ Store the filtrate -20 ℃.

**Note**
Weight the gel before adding PC solution, 0.1g gel approximately equal to 100μl solution.

**Infusion**
**Material**
5 × CE MultiS Buffer
Exnase MultiS
Double Distillation Water (ddH2O)
Primer
**Steps**
① Calculate the amount of DNA required for the recombination reaction according to the formula.

② Prepare the following reaction system on ice:
a. X/Y calculates the amount of vector and the amount of each insert according to the formula.

b. Negative control-1 can be used to confirm whether there is a circular plasmid residue in the
linearized cloning vector, and it is recommended.

c. Negative control-2 is recommended when the insert amplification template is a circular
plasmid with the same resistance as the cloning vector.

It is recommended that the circular plasmid residue detection of the linearized vector and
insert be performed independently.

d. Positive control reactions can be used to exclude the influence of other experimental
materials and operating factors.

③Use a pipette to gently mix (do not shake to mix), and centrifuge briefly to collect the reaction
solution to the bottom of the tube.

④React at 37°C for 30 min; drop to 4°C or cool on ice immediately.

**Note**

① In order to ensure the accuracy of sample addition, the linearized vector and insert can be
properly diluted before preparing the recombinant system, and the sample amount of each
component is not less than 1 μl.

②Calculation of vector and fragment usage:

Optimum usage per segment=(0.02×Fragment base pairs) ng (0.03 pmol)

For example, when the inserts with lengths of 0.5 kb, 1 kb, and 2 kb are cloned into a cloning
vector with a length of 5 kb, the optimal amount of the vector and each fragment to be used is:
Optimum use amount of linearized cloning vector: 0.02 × 5000 = 100 ng;

Optimum usage of 0.5 kb insert: 0.02 × 500 = 10 ng;

Optimum usage of 1 kb insert: 0.02 × 1000 = 20 ng;

Optimum use of 2 kb insert: 0.02 × 2000 = 40 ng

a. The amount of linearized cloning vector should be between 50 - 200 ng. When using the
above formula to calculate the optimal amount of DNA to be used beyond this range, simply
select the minimum/maximum amount of use.

b. The amount of each insert should be greater than 10 ng. When using the above formula to
calculate the optimum usage amount is lower than this value, just use 10 ng directly.

c. When the linearized cloning vector and the amplified product of the insert fragment are used
directly without DNA purification, the total volume added should not exceed 1/5 of the
volume of the reaction system, that is, 4 μl.

**Overlap PCR & Agarose Gel Electrophoresis**

**Material**

2 x Phanta Max Master Mix

Primer

Double Distillation Water (ddH2O)

Loading Buffer

DNA Marker

**Steps**

① Adding 25μl of 2 x Phanta Max Master Mix(5μM), 1μl per fragment, 4μl of primer
mix, then add ddH2O up to 50μl into a PCR tube. Different group use specific tube
with distinctive sign.

② Place those PCR tubes into Peltier thermal cycler.

③ Set the protocol as follow: begin at 95℃ for 30 secs, then keep 95℃ for 15 secs
for denaturation, decrease to 60℃ for 15 secs, 72℃ for 1 min/kb and repeat that
cycle 30 times, finally maintain 16℃ infinity.

④ Adding all the samples to the hole. Run the gel at 120V for about 25 minutes.
Check the result under the Blue Light Gel Imager.
**Note**

As we use Green Taq Mix, we don’t need to add Loading when Agarose Gel Electrophoresis.

**PCR & Agarose Gel Electrophoresis – Colony PCR**

**Material**

Green Taq Mix, Primer, Double Distillation Water (ddH2O), Eight tube centrifuge, EP Tube,
Tweezers, plastic wrap, Shaker, PCR Eight-Tube, DNA Marker

**Steps**

(Take 5 tubes of eight tube centrifuge for example)

1) Pick bacteria

① Add 1mL Amp LB to 1.5mL EP Tube.

② Use a small tip with tweezers to pick out a single clone and put them into the EP
Tube.

③ Put the EP tube on the Rank and wrap it with plastic wrap.

④ Insert it into the shaker obliquely, shake for 2h.

2) Colony PCR（after shaking for 2h）(take eight tubes for instance)

① Adding 6.6μl Primer Mix(5μl), 41.3μl 2 x Green Taq, 29.1μl of ddH2O to the first
tube of eight-tube, then draw 9ul from the first tube to the next 4 tubes.

② Centrifuge the eight-tube in a centrifuge for several seconds.

③ Take the eight-tube into Clean bench.

④ Add 1μl Bacteria Liquid.

⑤ Set the protocol as follow: begin at 95℃ for 3 mins, then keep 95℃ for 15 secs
for denaturation, decrease to 55℃ for 15 secs, 72℃ for 1 min and repeat that
cycle 30 times, 72℃ for 1 min, finally maintain 4℃ infinity.

⑥ Adding all the samples to the hole. Run the gel at 120V for about 25 minutes.

Check the result under the Blue Light Gel Imager.

⑦ Select the right System for Shaking bacteria and Plasmid Extraction.

**Note**

① The lid of the PCR Eight-tube is placed in the ultra-clean workbench to make sure
we add Bacteria Liquid at the ultra-clean workbench.

② As we use Green Taq Mix, we don’t need to add Loading when Agarose Gel
Electrophoresis.

**PCR & Agarose Gel Electrophoresis**

**Material**

2 x Phanta Max Master

Mix Primer

Double Distillation Water (ddH2O)

Loading Buffer DNA

Marker

**Steps**

① Adding 25μl of 2 x Phanta Max Master Mix, ណng Template DNA, 4μl of primer
mix, then add ddH2O up to 50μl into a PCR tube. Different group use specific tube
with distinctive sign.

② Place those PCR tubes into Peltier thermal cycler.

③ Set the protocol as follow: begin at 95℃ for 30 secs, then keep 95℃ for 15 secs
for denaturation, decrease to 60℃ for 15 secs, 72℃ for 1 min/kb and repeat that
cycle 30 times, finally maintain 16℃ infinity.

④ Adding all the samples to the hole. Run the gel at 120V for about 25 minutes.
Check the result under the Blue Light Gel Imager.

**Plasmid conservation**

**Material**

Cryopreservation tube

50% Glycerin

Alcohol lamp

**Steps**

① Add 400μl 50% Glycerin and 600μl bacterial liquid

② Mix it upside down and put it in the refrigerator at -80 degrees

**Note**

Burn the mouth and cap of the flask with an alcohol lamp before and after opening the
glycerin bottle.

**Plasmid Extraction**

**Material**

TIANprep Mini Plasmid Kit II

Absolute Ethanol

Double Distillation Water (ddH2O)

Centrifuge

**Steps**

① Insert the adsorption column into a collection tube, add 500μl BL equilibrium solution,
centrifuge at 12,000G for 1min, discard the filtrate and reuse the collection tube.

② Add the bacterium liquid to a centrifuge tube, centrifuge at 12,000G for 1min, remove
the supernatant as much as possible, keep the precipitate in the tube.

③ Add 500μl solution P1 (the RNase A need to be added in advance) into the centrifuge
tube. Use a pipette or vortex oscillator to completely suspend the bacterial cell
precipitate.

④ Add 500μl solution P2 into the centrifuge tube and turn it up and down gently for 6-8
times till the cells are fully lysed, when the liquid should be clear and sticky, meanwhile
12,000G for 10min.

⑥ Add the supernatant collected in the previous step into the adsorption column (the
adsorption column is put into the collection tube). Centrifuge at 12,000G for 1min,
discard the filtrate and reuse the collection tube.

⑦ Add 500μl solution PD to the adsorption column. Centrifuge at 12,000G for 1min,
discard the filtrate and reuse the collection tube.

⑧ Add 600μl solution PW (the absolute ethanol need to be added in advance) to the
adsorption column. Centrifuge at 12,000G for 1min, discard the filtrate and reuse the
collection tube.

⑨ Repeat step⑧.

⑩ Centrifuge the adsorption with collection tube at 12,000G for 2min, discard the filtrate
and reuse the collection tube. Open the cover of adsorption column and dry it out in the
air for 5min.

⑪ Place the adsorption column into a clean centrifuge tube. Add 200μl ddH2O to the
middle part of the adsorption membrane, placed it at room temperature for 2-5 min,
and centrifuge at 12,000G for 2min. The plasmid solution was collected into the

centrifuge tube.

**Note**

① Add RNase A to solution P1 before using.

② After adding solution P2, do not shake the tube violently to avoid pollution.

③ In step⑥, pay attention not to draw the white precipitate in the centrifuge tube out.

④ Add absolute ethanol to solution PW before using.

⑤ In step⑪, in order to increase the recovery efficiency of plasmid, the solution obtained
in the centrifuge tube can be readded into the centrifugal adsorption column and repeat
step⑪ again.

**Protocol for LB medium preparation**

**Material**

LB Broth Agar Powder, Miller LB Broth Powder, Miller Ultrapure (UP) Water, Double

Distillation

Water (ddH2O), Ampicillin ·

**Steps**

(1) Making LB liquid medium

① Weigh 12.5g of LB Broth Powder.

② Add the powder into 500ml UP Water.

③ Autoclave entire bottle of LB medium under 121 Celsius degree for 20 minutes.

(2) Making LB solid medium

① Weigh 12g of LB Broth Agar Powder.

② Add the powder into 300ml UP Water.

③ Autoclave entire bottle of LB medium under 121 Celsius degree for 20 minutes.

④ Pour the medium into culture dishes in the ultraclean worktable.

(3) Making LB selective medium

① Weigh 12g of LB Broth Agar Powder.

② Add the powder into 300ml UP Water.

③ Autoclave entire bottle of LB medium under 121 Celsius degree for 20 minutes.

④ Weigh 0.98g of Ampicillin.

⑤ Add the ampicillin into 9.8ml ddH2O. The ddH2O is sterilized previously. Shake the

mixture fully.

⑥ Filter the mixture through a filter to remove microorganism.

⑦ Add 1μl of mixture into an EP tube with 999μl ddH2O. The ddH2O is sterilized previously.

⑧ When the LB medium cool down to nearly 50℃, add 300μl antibiotic mixture into it in
the ultraclean worktable, then pour it into culture dishes

**Transformation**

**Material**

Competent Cell

Spreader

Plasmid (with target gene)

Ice Maker

Water Bath

Shaker

Oven

LB Liquid Medium

LB Selective Medium

**Steps**

① Take 100μl competent cell suspension from -80 ℃ refrigerator into an EP tube and put
it in ice bath until it thaws.

② Add 1μl plasmid with target gene into the suspension and put it in ice bath for 30min.

③ Put the EP tube into 42 ℃ water bath for 90sec.

④ Transfer the tube into ice bath for 3min immediately.

⑤ Add 900μl LB liquid medium into the tube, then shake it with 300rpm at 37 ℃ for 1h.

⑥ 5krpm, 5min Centrifuge

⑦ Discard the 850μl supernatant and resuspend the remaining 150μl

⑧ Apply 150μl bacterium liquid in a LB selective medium plate, then flip it and put it in

oven at 37℃ for 16h.

**Note**

① The whole process is operated under sterile condition.

② Switch on the ice maker and the water bath before beginning.

③ The time of step ③ and ④ must be controlled precisely.

### Agarose Gel Electrophoresis

### Double Enzyme DNA Digestion

### Gel Purification

### Infusion

### Overlap PCR & Agarose Gel Electrophoresis

### PCR & Agarose Gel Electrophoresis-Colony PCR

### PCR & Agarose Gel Electrophoresis

### Plasmid Conservation

### Plasmid Extraction

### Protocol for LB Medium Preparation

### Transformation

## Cell Experiments

**Protocol for Cell Transfection and Stimulation**
**Material**
96-well cell culture plates (or 24-well cell culture plates)
DMEM
Complete medium (High Glucose DMEM Medium supplemented with 10% FBS and 1% Penicillin Streptomycin Solution)
Polyethyleneimine (PEI) solution (1 mg/mL)
**Steps**
①Seed approximately $1×10^4$ cells into 96-well cell culture plates ($5×10^4$ for 24-well cell culture plates).
②Culture for 16h before transfection.
③Total plasmid mixes of 100ng per well (500ng for 24-well cell culture plates) are mixed thoroughly in serum-free DMEM before PEI solution is added into the plasmid mixture in a ratio of 1:3 (plasmid weight/PEI weight).
④The plasmid - PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for 6-8 hours.
⑤Cells are changed into fresh medium and applied with stimulus before sampling and analysis assay.
**Note**
To validate and optimize the gene circuits to reach the intended functionality and performance with high throughput, transient transfections are preferred. Also, easily detected reporters (such as SEAP and Luciferase) are also preferred during this process to evaluate the dynamics of the circuits.

**Protocol for Cell Culture and Subculture**
**Material**
PBS
0.25% Trypsin EDTA solution
Complete medium (High Glucose DMEM Medium supplemented with 10% FBS and 1% Penicillin Streptomycin Solution)
**Steps**
(1)Cell culture
①Culture in a humidified incubator at 37°C, 5%CO2.
②Check the color medium; healthy usually leaves medium slightly orange; observe the cells under a phase microscope.
③If the cells are not very confluent, remove the growth medium and replace it with a fresh complete medium.
④If the cells are at least 70%-80% confluence, subculture them.
(2)Subculturing cells (60 mm × 15 mm cell culture plate)
①Aspirate the growth medium from the cells and wash the cells with 2ml PBS to clear the FBS.
②Aspirate the PBS, then add 1ml 0.25% trypsin EDTA solution.
③Incubation for several minutes at 37°C and check the culture with an inverted microscope to ensure the cells are rounded up, indicating they are detached from the surface.
④Add 1ml fresh complete medium, then pipet all the adherent cells into cell suspension and pipet up and down.
⑤Transfer the cell suspension into a 15ml centrifuge tube and centrifuge at 720g for 5 minutes.
⑥Aspirate the filtrate, then add 2ml fresh medium and resuspend the cell.
⑦Place 9.5 ml of fresh medium in each new culture. Add 500ul cell suspension to each of the fresh plates that have been appropriately labeled.
⑧Culture in a humidified incubator at 37°C, 5%CO2.
**Note**
The time of treating cells with 0.25% trypsin EDTA solution should not be too long. Otherwise, it will cause toxicity to cells and affect cell status

**Protocol for SEAP assay in vitro**
**Material**
Chemiluminescence-based SEAP assay kit (e.g., Sigma Aldrich; cat. no. 1177984200)
buffer (2x): 20 mM L-Homoarginine hydrochloride, 1 mM MgCl2, 21% (v/v)
diethanolamine, pH 9.8.
Substrate for SEAP: 120 mM para-nitrophenyl phosphate (pNPP) in 2SEAP buffer.
Transparent 96-well plates.
Colorimetric plate-reader.
**Steps**
① Sample 200 μL culture medium from each well, heat inactivate at 65 C for 30 min.
② During the heat inactivation procedure, warm up 2 SEAP buffer (100 μL/well) at
37 ℃.
③ Add 1/5 buffer volume of pNPP (20 μL/well) substrate into the 2x buffer to prepare
the “Detection Mixture.”
④ Add 80 μL heated medium into the 96-well plate, add 120 μL Detection Mixture.
⑤ Measure absorption at 405 nm, 30 s per read for 10 reads.
⑥ Calculate enzymatic activity.
**Note**
SEAP levels in culture supernatants can be quantified with a pnitrophenylphosphate-based
colorimetric assay.

**Protocol for Nluc assay in vitro**
**Material**
Nano-Glo Luciferase assay kit (e.g. Promega, Catalog number selected: N1120)
Nano-Glo Luciferase assay buffer.
Nano-Glo Luciferase assay substrate.
384-well plates
Colorimetric plate-reader.
**Steps**
① Sample 30 μL culture medium from each well
② Add buffer and substrate into 1.5mL centrifuge tube with ratio substrate : buffer = 1:50
③ Add 7.5 μL mixture of substrate and buffer into each well of the 384-well plate, add 7.5 μL medium into each well.
④ Measure absorption
⑤ Calculate enzymatic activity.
**Note**
NanoLuc levels in culture supernatants characterizes the activation intensity of the downstream signaling pathway of melatonin receptor.

### Protocol for cell transfection and stimulation

### Protocol for cell culture and subculture

### Protocol for SEAP assay in vitro

### Protocol for Nluc assay in vitro

{% endblock %}

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## Engineering of a synthetic gene circuit enabling melatonin-triggered transcriptional regulation in mammalian cells

Drug discovery would greatly benefit from a high-throughput drug screening platform that allows simultaneous assessment of multiple drug candidates with resource-efficient and simple experiments. To identify potential melatonin receptor agonists more efficiently, we designed a melatonin-regulated reporter assay, which utilizes a synthetic excitation-transcription circuit that couples the activation of melatonin receptor 1a (MTNR1A) with the expression of secreted Nanoluc luciferase (Nluc).

In mammals, the activation of the melatonin receptor is known to increase intracellular calcium levels by activating phospholipase C (PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). The resulting IP₃ stimulates the release of Ca²⁺ from the endoplasmic reticulum (ER) into the cytosol, increasing intracellular calcium concentrations 1. Building on this well-characterized signaling pathway, we designed a synthetic melatonin-responsive circuit incorporating MTNR1A and a chimeric calcium-sensing promoter (PNFAT). This promoter consists of multiple repeats of nuclear factor of activated T cells (NFAT) binding sites followed by a minimal CMV promoter. Elevating intracellular calcium levels would activate endogenous NFAT, leading to its binding at the NFAT sites and subsequent transcription of downstream proteins **(Fig. 1A)**.

To assess the functionality of this circuit, we co-transfected human embryonic kidney (HEK-293T) cells with MTNR1A-expressing pNC099 plasmids, alongside either pNC102, pNC100, pNC104, or pNC103, which encode a PNFAT-driven Nluc-expressing cassette. Unexpectedly, no significant Nluc expression was observed in cells treated with near-physiological concentrations of melatonin (1 nM) compared to untreated controls **(Fig. 1B)**. In contrast, robust Nluc expression could be detected in cells treated with Thapsigargin **(Fig. 1C)**, a known inducer of intracellular calcium release via ER depletion 2. In addition, cells transfected with pNC107 (lacking NFAT binding sites) showed no significant response to Thapsigargin, confirming the specificity of the response. These findings suggest that MTNR1A may not effectively induce sufficient calcium signaling in HEK-293T cells to activate the NFAT-dependent calcium-responsive promoters.

![Figure 1](https://static.igem.wiki/teams/5267/shuiyin/results-fig1.webp)

Figure 1 NFAT-based calcium-sensing promoters failed to response to the activation of melatonin receptor. (A) Schematic diagram of the gene circuit design that response to the MTNR1A-induced elevation of calcium level. The activation of MATNR1A triggers the elevation of intracellular Ca2+ level via the PLC/IP3 pathway; the elevated Ca2+ triggers the dephosphorylation of NFAT, which would then bind to the NFAT binding sites on the chimeric promoter and activate the downstream gene expression. (B) The PNFAT promoters respond poorly to melatonin receptor activation. HEK293T cells were co-transfected with melatonin receptor plasmid pNC099(PCMV-MTNR1A) and either pNC102(PNFAT\_1-IgK-Nluc), pNC100(PNFAT\_5-IgK-Nluc), pNC104(PNFAT\_6-IgK-Nluc) or pNC103(PNFAT\_7-IgK-Nluc). Cells were treated with either DMSO or melatonin 6 hours post transcription. Data represents mean±SD of nanoluc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (C) The PNFAT promoters respond robustly to the Thapsigargin-induced elevation of intracellular calcium level. HEK293T cells were transfected with either pNC102(PNFAT\_1-IgK-Nluc), pNC100(PNFAT\_5-IgK-Nluc), pNC104(PNFAT\_6-IgK-Nluc) or pNC103(PNFAT\_7-IgK-Nluc). Cells were treated with either DMSO or Thapsigargin 6 hours post transcription. Data are mean±SD of nanoluc expression levels measured 48 h after thapsigargin stimulation (n = 3 independent experiments).

## The reason why the pathway doesn't work - the up-regulation of Ca2+ is transient

To investigate why the calcium-based circuit did not perform as expected, we consulted Dr. Yiming Dong, a synthetic biologist, and Chief Scientific Officer of Atantares. Drawing from her experience with calcium-sensing promoters in mammalian cells, Dr. Dong suggested that we examine the calcium dynamics triggered by melatonin receptors. She noted that, in her experience, most calcium-responsive promoters required a sustained elevation of intracellular calcium levels for proper activation (refer to the Human Practices and Engineering section for more details).

To assess the calcium dynamics following MTNR1A activation, we co-transfected HEK-293T cells with the MTNR1A-expressing pNC099 plasmid and a calcium indicator plasmid encoding GCaMP6 3 (PCMV-GCaMP, courtesy of Mingqi Xie' s lab at Westlake University). Surprisingly, we observed no significant change in GCaMP fluorescence in cells treated with melatonin for 48 hours, either through fluorescence microscopy **(Fig. 2A)** or with a fluorescent plate reader **(Fig. 2B)**, compared to untreated controls. However, real-time live-cell calcium imaging revealed a significant spike in intracellular calcium levels immediately after melatonin administration **(Fig. 2C)**. Importantly, this calcium surge was short-lived, dissipating within 100 seconds of melatonin stimulation. In contrast, Thapsigargin induced a prolonged elevation of intracellular calcium levels **(Fig. 2C)**.

These findings suggest that the transient nature of melatonin-induced calcium elevation is likely the primary reason for the lack of responsiveness in our PNFAT-Nluc design. Developing a new gene switch that responds to such rapid calcium fluctuations would be time-consuming, prompting us to consider exploring alternative signaling pathways.

![Figure 2](https://static.igem.wiki/teams/5267/shuiyin/results-fig2.webp)

Figure 2.
Melatonin-triggered alteration of intracellular Calcium dynamics in HEK-293T cells. (A and B) Long-term calcium response to melatonin stimulation in HEK-293T cells. HEK293T cells were co-transfected with the MTNR1A-expressing plasmid pNC099 (PCMV-MTNR1A) and the plasmid expressing the calcium indicator GCaMP6 (PCMV-GCaMP). Cells were treated with either DMSO or Melatonin 6 hours post transfection. For (A), Fluorescent images were taken 48 h after stimulation, scale bar: 100 µm. Data are representative images of 3 independent experiments. For (B), cells were subjected to fluorescence detection with a plate reader 48 h after stimulation. Data are mean±SD of relative fluorescence intensity (in RFU) measured 48 h after stimulation (n = 8 independent experiments). (C) Short-term calcium response to melatonin stimulation in HEK-293T cells. HEK293T cells were co-transfected with the MTNR1A-expressing plasmid pNC099 (PCMV-MTNR1A) and the plasmid expressing the calcium indicator GCaMP6 (PCMV-GCaMP) and treated with the indicated reagent at the 48 h post transfection. Cells were imaged through a fluorescent microscope every 5 seconds, and 20 cells were selected for fluorescent analysis in each group with ImageJ software. The cellular fluorescent levels of each group were normalized to the steady state level before chemical induction, Data shows Mean±SEM.

## Successfully set up a platform for MTNR1A drug screening through the cAMP pathway and validated the feasibility

We then looked at other potential downstream signaling pathways of MTNR1A that we may possibly build synthetic gene circuits. Interestingly, we came across a very recent research that showed a robust increase of cAMP levels in melatonin-treated, MTNR1A-expressing HEK-293T cells 4. Additionally, other research has shown increased phosphorylation of cAMP-response element binding protein (CREB) in melatonin-stimulated cells 5. Following these leads, we proposed the possibility of rewiring the activation of MTNR1A to target gene transcription via the cAMP/PKA/CREB pathway. To accomplish this, we developed a new synthetic melatonin-responsive circuit, which includes MTNR1A and a chimeric cAMP-sensing promoter (PCRE) containing multiple repeats of CREB binding sites followed by a miniCMV promoter. Activation of MTNR1A would lead to an increase in cAMP and the phosphorylation of the endogenous CREB protein. The phosphorylated CREB protein would then bind to the CREB site of the PCRE promoter, activating the expression of the downstream genes **(Fig. 3A)**.

To evaluate the function of this circuit, we co-transfected the HEK-293T cells with MTNR1A-expressing pNC099 plasmids and either pNC101, pNC105, or pNC106, which encodes PCRE-driven Nluc-expressing cassette. As expected, cells expressing MTNR1A and PCRE-Nluc variants showed significant activation of Nluc expression upon melatonin treatment compared to the untreated control. Among these PCRE variants, the variant contains 4 tandem repeats of the CRE sequence showed the optimal performance (~8.62-fold activation compared to the untreated control, **Fig. 3B**). Also, fine-tuning the PCRE-Nluc level could efficiently adjust the basal expression level and the dynamic range of the system **(Fig. 3C)**.

To further characterize the melatonin-activating circuit, we generated a stable HEKMT cell line carrying both PCRE4-Nluc and PCMV- MTNR1A - expressing cassette with the sleeping beauty transpose system. As shown in **Fig. 3D**, HEKMT cells exposed to a gradient amount of melatonin could produce detectable amount of Nluc at around the 12th hour in a dose-dependent manner, with a EC50 of 0.4228 nM at the 24 h post melatonin stimulus **(Fig. 3E)**. Additionally, we also showed that HEKMT cells are also capable of responding dose-dependently to Ramelteon, a commercially available melatonin receptor agonist **(Fig. 3F)**. Together, these findings demonstrated the feasibility of the HEKMT cells as the basis of a drug-screening platform.

![Figure 3](https://static.igem.wiki/teams/5267/shuiyin/results-fig3.webp)

Figure 3.
The construction and characterization of a cAMP/CREB-based melatonin-responsive gene circuit in HEK-293T cells. (A) Schematic diagram of the gene circuit design that responds to the MTNR1A-induced alteration of cAMP level. The activation of MATNR1A triggers the elevation of intracellular cAMP level, which triggers the activation of CREB, which would then bind to the CRE sites on the chimeric promoter and activate the downstream gene expression. (B) Co-expression of MTNR1A and PCRE-promoter variants enables robust transcriptional activation upon melatonin stimulation. HEK293T cells were co-transfected with pNC099(PCMV-MTNR1A) and either pNC101(PCRE\_4-IgK-Nluc), pNC105(PCRE\_5-IgK-Nluc), or pNC106(PCRE\_6-IgK-Nluc). Cells were treated with DMSO or melatonin at 6 hours post transfection; data are mean±SD of nanoluc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments). (C) Finetuning of PCRE4-Nluc allows the tuning of system behavior. HEK293T cells were co-transfected with pNC099(PCMV-MTNR1A) and pNC101(PCRE\_4-IgK-Nluc) under the indicated plasmid ratio. Cells were treated with DMSO or melatonin at 6 hours post transfection. Data are mean±SD of nanoluc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (D) Step-response dynamics of HEKMT cells under melatonin treatment. HEKMT cell line carrying both PCRE4-Nluc and PCMV-MTNR1A expressing cassette was stimulated with the indicated amount of melatonin 16 hours post seeding. Nanoluc expression levels were measured at different time points after melatonin stimulation (Data are mean±SD, n = 3 independent experiments). (E) Dose-dependence curve of HEKMT cells under melatonin treatment. HEKMT cell line was stimulated with the indicated amount of melatonin 16 hours post seeding. Nanoluc expression levels were measured at 24 h after melatonin stimulation (Data are mean±SD, n = 3 independent experiments, EC50=0.4228 nM). (F) Ramelteon response of the HEKMT cells. HEKMT cells were treated with ramelteon 16 h post seeding. Nanoluc expression levels were measured at 24 h after ramelteon stimulation (Data are mean±SD, n = 3 independent experiments).

## Designer cell-based high throughput screen of plant essential oils to identify MTNR1A agonism

With a functioning melatonin receptor-responsive gene circuit, we then went on to build a drug screening platform to help screening potential MTNR1A agonists in a high throughput manner. On the basis of the HEKMT cells, we generated a control cell line stably expressing only the PCRE4-Nluc cassette (HEKCTR cells) so we can distinguish the MTNR1A-independent activation of the PCRE4-Nluc cassette from the real MTNR1A agonism reported by the HEKMT cells. While we were building the HEKCTR cells, we interviewed the front-line psychiatrists and engaged the potential user of the drug we might discover through our screening platform to gain some hints about what we can start testing our screening platform on. Interestingly, the term “aromatherapy” pops up as its been widely used around the globe for thousands of years to help falling asleep, and the general public feels more comfortable using these natural product as a daily supplement (See Human Practices and Engineering page for more information). Hence, we purchased 37 plant essential oils online and hope to see if we can get lucky finding some new agonists for MTNR1A.

Intriguingly, while most of the plant essential oils showed no significant alteration in the Nluc production of either the HEKMT cells or the HEKCTR cells, we surprisingly found that the HEKMT cells treated with EO016 oil showed a robust elevation in Nluc production compared to the DMSO-treated control (~5.25-fold). In comparison, Nluc production in HEKCTR cells showed no significant alteration, suggesting that EO016 oil activates Nluc production in a MTNR1A-dependent manner **(Fig. 4a)**. Similar results were also observed on EO017 Oil, with a milder induction rate of Nluc production in HEKMT cells (~2.32-fold, **Fig. 4a**). Further analysis revealed that both EO016 oil and EO017 Oil could robustly induce MTNR1A-dependent Nluc production, but no clear dose-dependence could be observed on EO017 Oil **(Fig. 4b and 4c)**. These findings not only showed the feasibility of our designer-cell-based platform for rapid, robust, and resource-efficient high-throughput drug screening but also identified two essential oil hits for agonist discovery.

![Figure 4](https://static.igem.wiki/teams/5267/engi-fig8-bianhao.webp)

Figure 4.
Designer cell-based high throughput screen of plant essential oils to identify MTNR1A agonism. (A) High-throughput analysis. HEKCTR refers to stable HEK293T cells stabling expressing PCRE\_4-Nluc, HEKMT refers to the HEK293T cells shown in **Fig. 3D-3F**. Cells were treated with 1×104 v/v of either DMSO or indicated essential oil 16 h post seeding. Cells treated with 1 nM melatonin were used as positive control. Nanoluc expression levels were measured at 24 h after exposure to essential oils (Data are mean±SD, n = 3 independent experiments). (B, C) Dose-dependent validation of the most active essential oil hits. (B) HEKCTR and (C) HEKMT were treated with different essential oil dilutions (v/v) 16 h post seeding. Nanoluc expression levels were measured at 24 h after exposure to essential oils (Data are mean±SD, n = 3 independent experiments).

## Integrating in silico virtual screening and cell-based in vitro screening enables the discovery of MO001 as a potential MTNR1A-agonist

Most essential oils are a mixture of multiple natural product molecules contributing differently to the overall therapeutic function. However, screening over all constituents in the essential oil hits to identify the functioning molecule can sometimes be difficult due to the availability of the constituents and the time- or financial-constraints. Similar concerns were also raised by Dr. Mingshun Li, the instructors of HZAU-CHINA, during our presentation in the Central China iGEMer’s Meetup in Wuhan, China. Dr. Li suggested us to screen over the essential oil constituents using in silico before moving towards the cellular experiments (refer to the Human Practice Page for more information). In addition, we also realized that by incorporating in silico screening against MTNR1A and MTNR1B, we may possibly find molecules that can selectively interact with MTNR1A instead of MTNR1B, therefore addressing the concerns raised by the clinical psychiatrists in our human practice efforts.

To identify the putative active constituents in the EO016 and EO017 essential oils that account for the activation of melatonin receptors, we generated an AutoDock Vina 6-based pipeline to predict the binding affinity of the essential oil constituents against the known structure of melatonin receptors (PDB IDs: 6ME2 for MTNR1A and 7VH0 for MTNR1B). The result suggest that CTL 01-05-B-A05 is a highly potent binder to both MTNR1A and MTNR1B (**Fig. 5A**, upper panel, also see the Modeling page for more information), outperforming other ligands in binding affinity. Notably, It displays a clear preference for MTNR1A over MTNR1B. In contrast, melatonin was found to be a relatively weak binder towards MTNR1A/MTNR1B receptors, consistent with its role in promoting sleep and being naturally degraded during the sleep cycle. These findings validate the feasibility of the docking parameters employed. Then, we performed in silico screening of all constituents of EO016 7 and EO017 8 essential oils by molecular docking. Interestingly, the major constituents of EO016 oil were found to bind to both MTNR1A/ MTNR1B receptors. Only MO001 exhibited a preference towards MTNR1A **(Fig. 5A)**. Structural analysis revealed that the hydroxy group of MO001 may form a hydrogen bond with the main chain oxgen of Ala104 in MTNR1A **(Fig. 5B)**. More importantly, in vitro assay showed a significantly increased Nluc production in HEKMT cells treated with MO001, while no Nluc induction was observed in HEKCTR cells **(Fig. 5C)**. Together, these results demonstrated the feasibility of our drug-screening pipeline that incorporates both designer cell-based in vitro screening and molecular docking-based in silico screening, and also suggested that MO001 might serve as a promising selective agonist for MTNR1A.

![Figure 5](https://static.igem.wiki/teams/5267/engi-fig10-bianhao.webp)

Figure 5.
Figure 5. In silico and in vitro screening of MTNR1A selective constituents from essential oils. (A) Heatmap of binding affinities of various melatonin receptor agonists and natural small molecules from EO016 and EO017 towards MTNR1A and MTNR1B. The main constituents’ ratios were presented within parentheses.
(B) Structural visualization of MO001 binds with MTNR1A (left panel). MTNR1A protein is shown as cartoon, displayed as rainbow colors by different helix bundle. MO001 molecule is shown as magenta sticks. 2D-interaction of MO001 with MTNR1A (right panel). Potential hydrogen bond is shown between MO001 and Ala104.
(C) Quantification of MTNR1A agonism by MO001 using designer cells. HEKCTR (left) and HEKMT (right) were treated with the indicated concentration of MO001 16 h post seeding. Nanoluc expression levels were measured at 24 h after MO001 induction (Data are mean±SD, n = 3 independent experiments).

## References

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Lap Hang Tse, S. T. C.,  Seayoung Lee, Yung Hou Wong. Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor. *Int. J. Mol. Sci.* **25**, 2919 (2024). https://doi.org/10.3390/ijms25052919

Kong, P.-J. et al. Melatonin Induces Akt Phosphorylation through Melatonin Receptor- and PI3K-Dependent Pathways in Primary Astrocytes. *Korean J.Physiol. Pharmacol.* **12**, 37-41 (2008). https://doi.org/10.4196/kjpp.2008.12.2.37

Eberhardt, J., Santos-Martins, D., Tillack, A. F. & Forli, S. AutoDock Vina 1.2.0: new docking methods, expanded force field, and python bindings. *J. Chem. Inf. Model.* **61**, 3891-3898 (2021). https://doi.org/10.1021/acs.jcim.1c00203

Komori, T., Matsumoto, T., Motomura, E. & Shiroyama, T. Sleep-enhancing effect of valerian inhalation and sleep-shortening effect of EO028 inhalation. *Chem Senses.* **31**, 731-737 (2006). https://doi.org/10.1

Mohammadkhani, P. G., Irandoust, K., Taheri, M., Mirmoezzi, M. & Baić, M. Effects of eight weeks of aerobic exercise and taking EO017 supplement on C-reactive protein and sleep quality in obese women. *Biological Rhythm Research* **52**, 218-226 (2021). https://doi.org/10.1080/09291016.2019.1587837

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Results

[Ca2+ pathway report failure](#block1)

[Transient up-regulation of Ca2+](#block2)

[Platform building for MTNR1A-cAMP pathway](#block3)

[Preliminary screening of essential oils](#block4)

[In silico and in vitro screening](#block5)

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