In mammalian cell synthetic biology, utilizing synthetic or endogenous biological signaling pathways is critical for repurposing them for customized and programmable novel functions. A platform that can sense and characterize the status (such as activation/inhibition) of these biological pathways can provide valuable insights into their functionality and offer toolkits for potential biotechnological applications, such as cell-based high-throughput drug screening, as demonstrated in our project.
To this end, we present a collection of parts that can be assembled into distinct cell-based screening platforms, which can accurately sense and characterize the status of three important signaling pathways in mammalian cells: the cAMP/PKA/CREB pathway, the MAPK/ERK pathway, and the Ca2+ signaling pathway. Below, we demonstrate how these cell-based pathway-responsive screening platforms can be constructed using our parts collection and test their functionalities with detectable reporter signals.
The cAMP signaling pathway is crucial for various cellular processes, including the regulation of metabolism, gene expression, and cell growth1. To construct a cAMP/PKA/CREB signaling pathway-responsive characterization platform base on mammalian cell chassis, we can use 4×CRE, 5×CRE and 6×CRE respectively (BBa_K5267040, BBa_K5267041, BBa_K5267042) , together with Pmin-IgK->Nluc->bGH polyA, to build a synthetic gene circuit that senses specific stimuli and produces a measurable output.
The following is an example of building a cAMP/PKA/CREB signaling pathway characterization platform. We use this platform to characterize the activation of the cAMP/PKA/CREB signaling pathway upon stimulation with Forskolin.
Forskolin, a known activator of adenylate cyclase (AC), is used to stimulate the cAMP signaling pathway and measure the resulting expression of the reporter gene2. We used forskolin as a stimulant and constructed plasmids carrying the reporter gene NanoLuc and different CRE copies (4×CRE, 5×CRE, and 6×CRE; BBa_K5267040, BBa_K5267041, BBa_K5267042), such as 4xCRE_Pmin-IgK->Nluc->bGH_polyA (BBa_K5267040). These plasmids were co-transfected with PCMV->MTNR1A->bGH_polyA (BBa_K5267047) into HEK293T cells, which were then stimulated with forskolin.
After 48 hours, we measured the expression of NanoLuc in different platforms. The results confirmed that the cAMP/PKA/CREB signaling pathway was activated by forskolin as expected, with platforms with different copies of CRE showing different NanoLuc expression fold changes. This demonstrated the correct responsiveness of the cAMP signaling pathway (Figure 2).
Figure 1 : The expression of NanoLuc in different cell-based platforms stimulated by forsklin for 48 hours.
Calcium ions (Ca2+) impact nearly every aspect of mammalian cellular life, influencing biological processes such as excitability, exocytosis, motility, apoptosis, and transcription3. To accurately assess the status of Ca2+ signaling pathway in mammalian cell cytosol, we can construct platforms for characterization the Ca2+ signaling pathway using parts 1×NFAT, 5×NFAT, 6×NFAT, 7×NFAT and GCaMP (BBa_K5267007, BBa_K5267008, BBa_K5267009, BBa_K5267010, BBa_K3755007). Below are examples of how we used this platform to characterize the Ca2+ signaling pathway.
We constructed plasmids carrying synthetic promoter Pmin_NFAT with different copy numbers of NFAT (1×, 5×, 6×, 7×, respectively), and co-transfected these plasmids with PCMV->MTNR1A->bGH_polyA (BBa_K5267047) into HEK293T cells, this combination forms a synthetic genetic circuit that allows an accurate characterization of Cytosol Ca2+ concentration. We then stimulate cells with thapsigargin, a well-established inducer of intracellular calcium release via ER depletion, to detect the expression level of the reporter gene Nanoluc.
The results indicate that these parts in Ca2+ signaling pathway characterization platform can indirectly reflect the change in Ca2+ intracellular calcium release induced by thapsigargin.
Figure 2 : 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).
GCaMP (BBa_K3755007) can sense changes in calcium ion concentration intracellularly and emit green fluorescence. This provides us with a convenient tool for characterizing the downstream Ca2+ signaling pathway of mammalian cell GPCRs.
We utilized PCMV->GCaMP (BBa_K3755007) carrying GCaMP (BBa_K3755007) for real-time detection of intracellular Ca2+ concentration. We transfected PCMV->GCaMP (BBa_K3755007) into cells and detected the real-time live-cell fluorescence intensity under thapsigargin stimulation.
Under the stimulation of thapsigargin, real-time live cell fluorescence intensity of Ca2+ showed a significantly surge within 100 seconds and continues to respond thereafter (Figure 3).
Figure 3 : Real-time Ca2+ fluorescence intensity of different groups of cells after the stimulation of melatonin or thapsigargin.
MAPK-ERK signaling pathway is closely related to cell proliferation, differentiation, migration and tumorigenesis4. To sense and characterize the MAPK-ERK signaling pathway in mammalian cells, we developed MAPK-ERK signaling pathway characterization platform using TetR-ELK1(BBa_K3734019) and TCE(BBa_K4016011), Below, we describe how we use this platform to characterize the changes in the MAPK-ERK pathway under insulin stimulation.
When the insulin receptor (INRS) is stimulated by insulin, it can pass the signal and ultimately activate MAPK. Previous studies have coupled TetR-ELK1 with the MAPK-ERK signaling pathway to sense insulin stimulation5. We constructed three plasmids utilizing EF1a-INSR, TetR-ELK1, and TCE-SEAP respectively and co-transfected these plasmids into HEK293T cells.
The results indicate that the MAPK-ERK signaling pathway characterization platform can characterize changes in the MAPK-ERK signaling pathway under insulin stimulation (Figure 4).
Figure 4 : Functional validation of TetR-ELK1 pathway under insulin stimulation. HEK293T cells were transfected with P_EFla-INRSTetR-ElKl and TCE-SEAP in a 1:1:1 ratio and stimulated with insulin at concentrations of 1nm, 10nm, and 20nm after 24 hours to detect the SEAP activity, data shows mean±SD, n=3 independent experiments.
| Part Number | Name | Function |
| BBa_K5267004 | Pmin_4×CRE promoter | Function test of cAMP signaling pathway downstream of GPCR |
| BBa_K5267005 | Pmin_5*CRE promoter | |
| BBa_K5267006 | Pmin_6*CRE promoter | |
| BBa_K3734019 | TetR-ELk1 | Function test of ERK signaling pathway downstream of GPCR |
| BBa_K4016011 | TCE | |
| BBa_K5267007 | Pmin_1*NFAT promoter | Function test of Ca2+ signaling pathway downstream of GPCR |
| BBa_K5267008 | Pmin_5*NFAT promoter | |
| BBa_K5267009 | Pmin_6*NFAT promoter | |
| BBa_K5267010 | Pmin_7*NFAT promoter | |
| BBa_K3755007 | GCaMP6m |
Melatonin receptor structure and signaling. J. Pineal Res. 76, e12952 (2024).
Tordjman, S. et al. Melatonin: Pharmacology, Functions and Therapeutic Benefits. Curr. Neuropharmacol. 15, 434–443 (2017).
Gao, Y., Zhao, S., Zhang, Y. & Zhang, Q. Melatonin Receptors: A Key Mediator in Animal Reproduction. Vet. Sci. 9, 309 (2022).
Bagur, R. & Hajnóczky, G. Intracellular Ca2+ Sensing: Its Role in Calcium Homeostasis and Signaling. Mol. Cell 66, 780–788 (2017).
Le Bras, A. A bright fluorescent protein for neuronal imaging. Lab Anim. 50, 14–14 (2021).
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