Difference between revisions of "Part:BBa K5267040"

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	<partinfo>BBa_K5267040 short</partinfo>		<partinfo>BBa_K5267040 short</partinfo>
−	When melatonin binds to the MT1 receptor, the activation of adenylate cyclase (AC) is activated, regulating the level of the second messenger cAMP, while activating protein kinase to further amplify the signal. It catalyzes the phosphorylation of CREB in the nucleus and regulates the expression of downstream genes.Then the cell will glow blue fluorescence.	+	P_4×CRE->IgK->Nluc->bGH_polyA
		+
		+	<p>The cAMP/PKA/CREB pathway is a well-established signaling pathway in mammalian cells, regulated by various upstream pathways such as G-protein-coupled receptors (GPCRs). Upon activation of GPCRs, the cAMP/PKA/CREB pathways are triggered, ultimately leading to the phosphorylation of the endogenous transcription factor CREB. This phosphorylation allows CREB to bind to cAMP response elements (CRE) and regulate the expression of downstream genes.</p>
		+	<p>The composite part P_4×CRE->IgK->Nluc->bGH_polyA ([https://parts.igem.org/Part:BBa_K5267040 Part:BBa K5267040]) is designed to sense and characterize the status of the cAMP/PKA/CREB pathway based on its signaling transduction mechanism. Activation of this pathway results in increased phosphorylation of CREB, which then binds to four tandem repeats of the CRE site within the P_4CRE promoter, activating the expression of the reporter gene NanoLuc luciferase.</p>
		+
		+	<!-- -->
		+	==Sequence and Features==
		+	<partinfo>BBa_K5267040 SequenceAndFeatures</partinfo>
		+	==Profile==
		+	Name:P_4×CRE->IgK->Nluc->bGH_polyA
		+	<br>Base Pairs: 944bp
		+	<br>Origin: Homo sapiens
		+	<br>Properties:Enables sensing and characterizing the status (such as activation/inhibition) of the cAMP/PKA/CREB pathway
		+	==Usage and Biology==
		+	<p>'''G-protein-coupled receptors (GPCRs) '''represent the largest and most diverse group of membrane receptors in eukaryotes. G proteins are specialized proteins that can bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). When a ligand binds to a GPCR, it induces a conformational change in the receptor, which subsequently activates the associated G protein. The active form of the G protein is released from the receptor and dissociates into its subunits. These subunits then activate specific effectors, leading to the release of second messengers that are recognized by protein kinases, triggering a signaling cascade that results in various cellular events.[1] </p>
		+	<p>'''Cyclic adenosine monophosphate (cAMP)''' serves as a pivotal second messenger that transduces extracellular signals to intracellular effectors by modulating its concentration within the cell. Upon ligand binding to GPCRs, adenylate cyclase is activated via the Gα subunit, resulting in elevated levels of intracellular cAMP. Subsequently, cAMP interacts with the regulatory subunits of protein kinase A (PKA), leading to the dissociation of its catalytic subunits. The liberated catalytic subunits of PKA translocate to the nucleus, where they phosphorylate the transcription factor cAMP response element-binding protein (CREB). Phosphorylated CREB then binds to the coactivator CREB-binding protein (CBP), forming a complex that recognizes and binds to the cAMP response element (CRE) within the regulatory regions of target genes, thereby initiating transcriptional activation. [2] </p>
		+
		+	<p>This '''cAMP/PKA/CREB pathway '''is a well-established and common signaling pathway in mammalian cells, regulated by various importance upstream pathways such as GPCRs we describe above. Therefore, in the view of mammalian cell synthetic biology, a synthetic gene circuit that enables sensing and characterizing the status (such as activation/inhibition) of the cAMP/PKA/CREB pathway can be very useful that only provide valuable insights into their functionality and but also offer toolkits for potential biotechnological applications..</p>
		+	<p>To achieve the function described above, we present this composite that can directly sense and accurately characterize the status of the cAMP/PKA/CREB pathway in mammalian cells.</p>
		+
		+	<p>'''This composite part consisting of:'''</p>
		+	<p>'''1. P_4×CRE ([https://parts.igem.org/Part:BBa_K5267004 Part:BBa_K5267004])''': A synthetic promoter that assess the activation status of the cAMP/PKA/CREB signaling pathways, with 4 tandem repeats of CRE serving as a binding site for phosphorylated CREB.</p>
		+	<p>'''2. IgK ([https://parts.igem.org/Part: BBa_K3117006])''': A signal sequence directing the protein into the secretory pathway.</p>
		+	<p>'''3. Nluc ([https://parts.igem.org/Part: BBa_K2728003])''': An engineered luminescent reporter that detects cAMP concentration. When expressed, it results in blue fluorescence, indicating the activation status of the cAMP/PKA/CREB pathway.</p>
		+	<p>'''4. bGH_polyA ([https://parts.igem.org/Part: BBa_K1313006])''': Functions as a terminator, ensuring proper cessation of gene expression.</p>
		+	<p>By incorporating this composite part into mammalian cell chassis, such as the HEK293 cell line, we can build a cAMP/PKA/CREB pathway characterization platform that effectively senses and monitors the activation of the pathway, providing detectable reporter signals. This synthetic gene circuit can be a valuable tool for studying signaling pathways and can be applied in various biotechnological and therapeutic research areas.</p>
−	===Usage and Biology===
−	G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes. G proteins are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Ligand binding to the GPCR causes a change in the receptor conformation that in turn binds and activates the G-protein. The active form of the G-protein is then released from the surface of the receptor,  dissociating into its subunits. Both subunits will then activate their specific effectors, causing the release of second messengers. These messengers are recognised by protein kinases leading to their activation and triggering the signaling cascade towards a cellular event.[1]
−	Cyclic adenosine monophosphate (cAMP) serves as a pivotal second messenger that transduces extracellular signals to intracellular effectors by modulating its concentration within the cell. Upon ligand binding to G-protein coupled receptors (GPCRs), adenylate cyclase is activated via the Gα subunit, culminating in elevated levels of intracellular cAMP. Subsequently, cAMP interacts with the regulatory subunits of protein kinase A (PKA), precipitating the dissociation of its catalytic subunits. The liberated catalytic subunits of PKA translocate to the nucleus, where they phosphorylate the transcription factor cAMP response element-binding protein (CREB). Phosphorylated CREB then specifically binds to the coactivator cAMP response element-binding protein (CBP), forming a complex that recognizes and binds to the cAMP response element (CRE) within the regulatory regions of target genes, thereby initiating transcriptional activation.[2]
−	We hope to design a reporter plasmid vector for the melatonin receptor pathway and introduce it into the recipient cells for reporting。This part is based on the cAMP-CREB pathway with MT1 acting as a cell receptor (Fig.1). 4xCRE-Pmin (BBa_K5267004) is loaded into the vector  as a diagnostic element to assess the successful activation of the receptor's downstream signaling pathways., IgK (BBa_K3117006)is a signaling sequence, directing the protein into the secretory pathway , Nluc(BBa_K2728003) is engineered for optimal performance as a luminescent reporter to detect cAMP concentration [3]. If it is successfully expressed,  the cell will glow blue fluorescence. bGH_polyA (BBa_K1313006)is a terminator, controlling the cessation of gene expression .，
−	Based on the composition of this part, it functions as a cAMP concentration detection platform, and finally, this part is successfully delivered into HEK293 cell line and currently works properly when stimulated by melatonin.
	<html>		<html>
	<figure class="figure">		<figure class="figure">
−	<div style="width=100%;height=auto">	+	<div style="width=60%; height=60%; text-align: center">
−	< img src="https://static.igem.wiki/teams/5267/mao-parts/108bf7048666342015244d8e67a5ee3.png" class="figure-img img-fluid rounded"  height="400px">	+	<img src="https://static.igem.wiki/teams/5267/mao-parts/4cregaoqing.png"  height="100px">
−		+	</div>
	</figure>		</figure>
−
	</html>		</html>
−	Figure 1. Schematic of cAMP-CREB signal pathway. When melatonin binds to the MT1 receptor, the activation of adenylate cyclase (AC) is activated, regulating the level of the second messenger cAMP, while activating protein kinase to further amplify the signal. It catalyzes the phosphorylation of CREB in the nucleus and regulates the expression of downstream genes.	+	'''Figure 1. Schematic diagram of BBa_K5267040 composite part '''
−	===Special design===	+
−	This basic part is an important element for testing whether the downstream pathway of melatonin responds successfully. At present, the commonly used method to study the signaling pathway is to clone the response element of the transcription factor corresponding to the signaling pathway into the luciferase reporter gene vector, that is, pCRE-luc .[3] However, the expression effect of a single responder is weak，so multiple tandem repeats of the same responder element are usually inserted upstream of the reporter gene (the 5 '-UTR region) to enhance the activation of the signaling pathway. By searching through literature, we constructed the 4xCRE-Pmin sequence，It contains a 5′ minimal promoter incorporating 4 multimerized palin-dromic CREs with the sequence 5′-AGCC[TGACGTCC]GAG-3′. (CRE denoted in brackets)，with the exception of a single nucleotide substitution (C for A) within the CRE[4]，which may strengthen gene expression downstream.	+	==Special design==
−		+	The composite part P_4×CRE->IgK->Nluc->bGH_polyA ([https://parts.igem.org/Part: BBa_K5267040]) is essential for evaluating the responsiveness of the melatonin signaling pathway. Standard approaches for studying signaling pathways typically involve cloning the response element of the relevant transcription factor into a luciferase reporter gene vector, such as P<sub>cre</sub>-luc.[3] Due to the limited transcriptional impact of a single response element, we incorporated multiple tandem copies of the element near the reporter gene's genomic location. This amplification enhanced the initiation efficacy of the signaling cascade. Therefore, we constructed the P<sub>min</sub>_4×CRE sequence, which includes a 5′ minimal promoter incorporating 4 tandem repeats of CREs.[4] (Figure 2)
−	===Function Test===	+
−	==Method==	+
−	==Result==
	<html>		<html>
	<figure class="figure">		<figure class="figure">
−	<div style="width=100%;height=auto">	+	<div style="width=100%; height=auto; text-align: center">
−	< img src="https://static.igem.wiki/teams/5267/mao-parts/huge4xcre-f3.png" class="figure-img img-fluid rounded"  height="400px">	+	<img src="https://static.igem.wiki/teams/5267/mao-parts/gaoqing.png"  height="300px">
		+	</div>
		+	</figure>
		+	</html>
		+	'''Figure 2. Schematic of cAMP-CREB Signaling Transduction.''' When melatonin binds to the MT1, it activates adenylate cyclase (AC), which in turn regulates the level of the second messenger cAMP. The increase in cAMP activates protein kinase A (PKA), which amplifies the signal. PKA then catalyzes the phosphorylation of CREB in the nucleus, thereby binding to the P_4×CRE/P_5xCRE/P_6xCRE and initiate the transcription of Nluc
		+	==Function Test==
		+	===Method===
		+	<p>Forskolin, a known activator of adenylate cyclase (AC), is used to stimulate the cAMP signaling pathway2. 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 our composite part 4×CRE_Pmin-IgK->Nluc->bGH_polyA ([https://parts.igem.org/Part: BBa_K5267040]). These plasmids were co-transfected with PCMV->MTNR1A->bGH_polyA ([https://parts.igem.org/Part: BBa_K5267047]) into HEK293T cells. The cells were then stimulated with forskolin to determine whether forskolin-induced activation of the cAMP/PKA/CREB signaling pathway can be sensed and characterized by our composite part P_4×CRE -> IgK -> Nluc -> bGH_polyA.</p>
		+
		+	<p>To further validate and evaluate the function of the composite part P_4×CRE -> IgK -> Nluc -> bGH polyA (Part: BBa_K5267040), we co-transfected it with MTNR1A ([https://parts.igem.org/Part: BBa_K5267001]) into HEK-293T cell lines. This setup aims to mimic the melatonin receptor-mediated cAMP/PKA/CREB signaling transduction pathway. The melatonin-activated MTNR1A receptors should activate CREB, thereby binding to the 4×CREs and initiating NanoLuc expression from the composite part.</p>
		+	<p>Theoretically, upon stimulation with forskolin or melatonin, the cAMP/PKA/CREB signaling pathway is triggered, ultimately leading to the phosphorylation of the endogenous transcription factor CREB and activation of the P<sub>cre</sub>, thereby regulating the expression of downstream genes.</p>
		+
		+	===Result===
		+	<html>
		+	<figure class="figure">
		+	<div style="width=120%; height=auto; text-align: center">
		+	<img src="https://static.igem.wiki/teams/5267/mao-parts/4xcre.jpg"  height="280px">
		+	</div>
	</figure>		</figure>
		+	</html>
		+	'''Figure 3. The expression of NanoLuc in cAMP/PKA/CREB pathway-responsive cell platform stimulated by forskolin. '''
		+
		+	<p>We measured the expression of NanoLuc in both forskolin-treated and untreated groups 48 hours post-stimulation. The results confirmed that the cAMP/PKA/CREB signaling pathway was activated by forskolin as expected. Platforms with different tandem repeats of CRE exhibited varying fold changes in NanoLuc expression. Notably, cells transfected with P_4×CRE->IgK->Nluc->bGH polyA ([https://parts.igem.org/Part: BBa_K5267040]) showed the highest fold change (~8.62 fold) compared to the control. These results demonstrate the correct responsiveness of the cAMP signaling pathway '''(Figure 3)'''.</p>
		+	<html>
		+	<figure class="figure">
		+	<div style="width=120%; height=auto; text-align: center">
		+	<img src="https://static.igem.wiki/teams/5267/mao-parts/compart.png"  height="200px">
		+	</div>
		+	</figure>
	</html>		</html>
−	Figure 3. CREB activation in response to cAMP signaling. (a) Schematic diagram of MT1 receptor activating the downstream cAMP pathway. (b) Melatonin stimulation applied to HEK-293T cells co-transfected with pCJ008(PCMV-MTNR1A) and pNC005(PCRE_4-IgK-Nluc), pNC016(PCRE_5-IgK-Nluc), pNC017(PCRE_6-IgK-Nluc) respectively. Data are mean±SD of NanoLuc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments). (c) HEK-293T cells were co-transfected with pCJ008(PCMV-MTNR1A) and pNC005(PCRE_4-IgK-Nluc) under various transfection ratio(100:25/100:50/100:100/100:200). Data are mean±SD of NanoLuc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (d) Stable HEK-293T cells co-transfected with pCJ008(PCMV-MTNR1A) and pNC005(PCRE_4-IgK-Nluc) stimulated with various  concentrations of melatonin(0.1nM /0.5nM /0.7nM /1nM /10nM /30nM) . Data are mean±SD of NanoLuc expression levels measured at different time points
−	<!-- -->
−	<span class='h3bb'>Sequence and Features</span>
−	<partinfo>BBa_K5267030 SequenceAndFeatures</partinfo>
−	===reference===	+
		+	<p>'''Figure 4. Characterization of composite part P_4×CRE->IgK->Nluc->bGH_polyA. '''(A) Co-expression of MTNR1A and P<sub>cre</sub>-promoter variants enables robust transcriptional activation upon melatonin stimulation. Melatonin stimulation was added to HEK-293T cells co-transfected with PCMV-MTNR1A and P_4xCRE-IgK-Nluc, P_5×CRE-IgK-Nluc, P_6×CRE-IgK-Nluc respectively. (B) Co-expression of MTNR1A and P<sub>cre</sub>-promoter variants enables robust transcriptional activation upon melatonin stimulation. HEK-293T cells were co-transfected with PCMV-MTNR1A and P_4×CRE-IgK-Nluc under various transfection ratio (100:25/100:50/100:100/100:200). (C) Step-response dynamics of HEKMT cells under melatonin treatment. HEK-293T cell lines expressing PCMV-MTNR1A and P_4×CRE-IgK-Nluc was stimulated with various concentrations of melatonin (0.1nM /0.5nM /0.7nM /1nM /10nM /30nM) . Data are mean±SD of NanoLuc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments)</p>
		+
		+	<p>Moreover, we utilized melatonin receptor-mediated activation of the cAMP/PKA/CREB pathway as a strategy to test the functionalities of the composite part P_4×CRE->IgK->Nluc->bGH polyA. The results showed a significant increase in NanoLuc expression in the melatonin-stimulated group compared to the unstimulated group '''(Figure 4A and B)''', indicating that the composite part can correctly respond to melatonin stimulation as expected. Notably, the NanoLuc expression level in cells transfected with P_4×CRE -> IgK -> Nluc -> bGH polyA ([https://parts.igem.org/Part: BBa_K5267040]) was the highest among all experimental groups, with the highest fold change (~8.62-fold) compared to the control.</p>
		+
		+	<p>To further characterize this composite part, we generated a stable HEKMT cell line carrying both the P<sub>cre</sub>-Nluc and PCMV-MTNR1A expression cassettes using the Sleeping Beauty transposon system, as shown in '''(Figure 4C)'''. The results showed dose-dependent responses upon stimulation with different concentrations of melatonin. Therefore, we selected this composite part as the optimal component and constructed a cell-based screening platform based on P_4×CRE->IgK->Nluc->bGH polyA ([https://parts.igem.org/Part: BBa_K5267040]) for our iGEM project.</p>
		+
		+	==reference==
	[1] Q. Wang et al., "Structural basis of the ligand binding and signaling mechanism of melatonin receptors," Nat Commun, vol. 13, no. 1, p. 454, Jan 24 2022, doi: 10.1038/s41467-022-28111-3.		[1] Q. Wang et al., "Structural basis of the ligand binding and signaling mechanism of melatonin receptors," Nat Commun, vol. 13, no. 1, p. 454, Jan 24 2022, doi: 10.1038/s41467-022-28111-3.
−	<br>[2] A. J. Shaywitz and M. E. Greenberg, "CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals," Annu Rev Biochem, vol. 68, pp. 821-61, 1999, doi: 10.1146/annurev.biochem.68.1.821.	+	<p>[2] A. J. Shaywitz and M. E. Greenberg, "CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals," Annu Rev Biochem, vol. 68, pp. 821-61, 1999, doi: 10.1146/annurev.biochem.68.1.821.</p>
−	<br>[3] C. Kemmer, D. A. Fluri, U. Witschi, A. Passeraub, A. Gutzwiller, and M. Fussenegger, "A designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms," J Control Release, vol. 150, no. 1, pp. 23-9, Feb 28 2011, doi: 10.1016/j.jconrel.2010.11.016.	+	<p>[3] C. Kemmer, D. A. Fluri, U. Witschi, A. Passeraub, A. Gutzwiller, and M. Fussenegger, "A designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms," J Control Release, vol. 150, no. 1, pp. 23-9, Feb 28 2011, doi: 10.1016/j.jconrel.2010.11.016.</p>
−	<br>[4] O. G. Chepurny and G. G. Holz, "A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts," J Biomol Screen, vol. 12, no. 5, pp. 740-6, Aug 2007, doi: 10.1177/1087057107301856.	+	<p>[4] O. G. Chepurny and G. G. Holz, "A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts," J Biomol Screen, vol. 12, no. 5, pp. 740-6, Aug 2007, doi: 10.1177/1087057107301856.</p>
		+	<p>[5] H. H. Okamoto, E. Cecon, O. Nureki, S. Rivara, and R. Jockers, "Melatonin receptor structure and signaling," J Pineal Res, vol. 76, no. 3, p. e12952, Apr 2024, doi: 10.1111/jpi.12952.</p>

---

Latest revision as of 13:42, 2 October 2024

P_4xCRE->IgK->Nluc->bGH_polyA

P_4×CRE->IgK->Nluc->bGH_polyA

The cAMP/PKA/CREB pathway is a well-established signaling pathway in mammalian cells, regulated by various upstream pathways such as G-protein-coupled receptors (GPCRs). Upon activation of GPCRs, the cAMP/PKA/CREB pathways are triggered, ultimately leading to the phosphorylation of the endogenous transcription factor CREB. This phosphorylation allows CREB to bind to cAMP response elements (CRE) and regulate the expression of downstream genes.

The composite part P_4×CRE->IgK->Nluc->bGH_polyA (Part:BBa K5267040) is designed to sense and characterize the status of the cAMP/PKA/CREB pathway based on its signaling transduction mechanism. Activation of this pathway results in increased phosphorylation of CREB, which then binds to four tandem repeats of the CRE site within the P_4CRE promoter, activating the expression of the reporter gene NanoLuc luciferase.

Sequence and Features

Profile

Name:P_4×CRE->IgK->Nluc->bGH_polyA
Base Pairs: 944bp
Origin: Homo sapiens
Properties:Enables sensing and characterizing the status (such as activation/inhibition) of the cAMP/PKA/CREB pathway

Usage and Biology

G-protein-coupled receptors (GPCRs) represent the largest and most diverse group of membrane receptors in eukaryotes. G proteins are specialized proteins that can bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). When a ligand binds to a GPCR, it induces a conformational change in the receptor, which subsequently activates the associated G protein. The active form of the G protein is released from the receptor and dissociates into its subunits. These subunits then activate specific effectors, leading to the release of second messengers that are recognized by protein kinases, triggering a signaling cascade that results in various cellular events.[1]

Cyclic adenosine monophosphate (cAMP) serves as a pivotal second messenger that transduces extracellular signals to intracellular effectors by modulating its concentration within the cell. Upon ligand binding to GPCRs, adenylate cyclase is activated via the Gα subunit, resulting in elevated levels of intracellular cAMP. Subsequently, cAMP interacts with the regulatory subunits of protein kinase A (PKA), leading to the dissociation of its catalytic subunits. The liberated catalytic subunits of PKA translocate to the nucleus, where they phosphorylate the transcription factor cAMP response element-binding protein (CREB). Phosphorylated CREB then binds to the coactivator CREB-binding protein (CBP), forming a complex that recognizes and binds to the cAMP response element (CRE) within the regulatory regions of target genes, thereby initiating transcriptional activation. [2]

This cAMP/PKA/CREB pathway is a well-established and common signaling pathway in mammalian cells, regulated by various importance upstream pathways such as GPCRs we describe above. Therefore, in the view of mammalian cell synthetic biology, a synthetic gene circuit that enables sensing and characterizing the status (such as activation/inhibition) of the cAMP/PKA/CREB pathway can be very useful that only provide valuable insights into their functionality and but also offer toolkits for potential biotechnological applications..

To achieve the function described above, we present this composite that can directly sense and accurately characterize the status of the cAMP/PKA/CREB pathway in mammalian cells.

This composite part consisting of:

1. P_4×CRE (Part:BBa_K5267004): A synthetic promoter that assess the activation status of the cAMP/PKA/CREB signaling pathways, with 4 tandem repeats of CRE serving as a binding site for phosphorylated CREB.

2. IgK (BBa_K3117006): A signal sequence directing the protein into the secretory pathway.

3. Nluc (BBa_K2728003): An engineered luminescent reporter that detects cAMP concentration. When expressed, it results in blue fluorescence, indicating the activation status of the cAMP/PKA/CREB pathway.

4. bGH_polyA (BBa_K1313006): Functions as a terminator, ensuring proper cessation of gene expression.

By incorporating this composite part into mammalian cell chassis, such as the HEK293 cell line, we can build a cAMP/PKA/CREB pathway characterization platform that effectively senses and monitors the activation of the pathway, providing detectable reporter signals. This synthetic gene circuit can be a valuable tool for studying signaling pathways and can be applied in various biotechnological and therapeutic research areas.

Figure 1. Schematic diagram of BBa_K5267040 composite part

Special design

The composite part P_4×CRE->IgK->Nluc->bGH_polyA (BBa_K5267040) is essential for evaluating the responsiveness of the melatonin signaling pathway. Standard approaches for studying signaling pathways typically involve cloning the response element of the relevant transcription factor into a luciferase reporter gene vector, such as P_cre-luc.[3] Due to the limited transcriptional impact of a single response element, we incorporated multiple tandem copies of the element near the reporter gene's genomic location. This amplification enhanced the initiation efficacy of the signaling cascade. Therefore, we constructed the P_min_4×CRE sequence, which includes a 5′ minimal promoter incorporating 4 tandem repeats of CREs.[4] (Figure 2)

Figure 2. Schematic of cAMP-CREB Signaling Transduction. When melatonin binds to the MT1, it activates adenylate cyclase (AC), which in turn regulates the level of the second messenger cAMP. The increase in cAMP activates protein kinase A (PKA), which amplifies the signal. PKA then catalyzes the phosphorylation of CREB in the nucleus, thereby binding to the P_4×CRE/P_5xCRE/P_6xCRE and initiate the transcription of Nluc

Function Test

Method

Forskolin, a known activator of adenylate cyclase (AC), is used to stimulate the cAMP signaling pathway2. 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 our composite part 4×CRE_Pmin-IgK->Nluc->bGH_polyA (BBa_K5267040). These plasmids were co-transfected with PCMV->MTNR1A->bGH_polyA (BBa_K5267047) into HEK293T cells. The cells were then stimulated with forskolin to determine whether forskolin-induced activation of the cAMP/PKA/CREB signaling pathway can be sensed and characterized by our composite part P_4×CRE -> IgK -> Nluc -> bGH_polyA.

To further validate and evaluate the function of the composite part P_4×CRE -> IgK -> Nluc -> bGH polyA (Part: BBa_K5267040), we co-transfected it with MTNR1A (BBa_K5267001) into HEK-293T cell lines. This setup aims to mimic the melatonin receptor-mediated cAMP/PKA/CREB signaling transduction pathway. The melatonin-activated MTNR1A receptors should activate CREB, thereby binding to the 4×CREs and initiating NanoLuc expression from the composite part.

Theoretically, upon stimulation with forskolin or melatonin, the cAMP/PKA/CREB signaling pathway is triggered, ultimately leading to the phosphorylation of the endogenous transcription factor CREB and activation of the P_cre, thereby regulating the expression of downstream genes.

Result

Figure 3. The expression of NanoLuc in cAMP/PKA/CREB pathway-responsive cell platform stimulated by forskolin.

We measured the expression of NanoLuc in both forskolin-treated and untreated groups 48 hours post-stimulation. The results confirmed that the cAMP/PKA/CREB signaling pathway was activated by forskolin as expected. Platforms with different tandem repeats of CRE exhibited varying fold changes in NanoLuc expression. Notably, cells transfected with P_4×CRE->IgK->Nluc->bGH polyA (BBa_K5267040) showed the highest fold change (~8.62 fold) compared to the control. These results demonstrate the correct responsiveness of the cAMP signaling pathway (Figure 3).

Figure 4. Characterization of composite part P_4×CRE->IgK->Nluc->bGH_polyA. (A) Co-expression of MTNR1A and P_cre-promoter variants enables robust transcriptional activation upon melatonin stimulation. Melatonin stimulation was added to HEK-293T cells co-transfected with PCMV-MTNR1A and P_4xCRE-IgK-Nluc, P_5×CRE-IgK-Nluc, P_6×CRE-IgK-Nluc respectively. (B) Co-expression of MTNR1A and P_cre-promoter variants enables robust transcriptional activation upon melatonin stimulation. HEK-293T cells were co-transfected with PCMV-MTNR1A and P_4×CRE-IgK-Nluc under various transfection ratio (100:25/100:50/100:100/100:200). (C) Step-response dynamics of HEKMT cells under melatonin treatment. HEK-293T cell lines expressing PCMV-MTNR1A and P_4×CRE-IgK-Nluc was stimulated with various concentrations of melatonin (0.1nM /0.5nM /0.7nM /1nM /10nM /30nM) . Data are mean±SD of NanoLuc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments)

Moreover, we utilized melatonin receptor-mediated activation of the cAMP/PKA/CREB pathway as a strategy to test the functionalities of the composite part P_4×CRE->IgK->Nluc->bGH polyA. The results showed a significant increase in NanoLuc expression in the melatonin-stimulated group compared to the unstimulated group (Figure 4A and B), indicating that the composite part can correctly respond to melatonin stimulation as expected. Notably, the NanoLuc expression level in cells transfected with P_4×CRE -> IgK -> Nluc -> bGH polyA (BBa_K5267040) was the highest among all experimental groups, with the highest fold change (~8.62-fold) compared to the control.

To further characterize this composite part, we generated a stable HEKMT cell line carrying both the P_cre-Nluc and PCMV-MTNR1A expression cassettes using the Sleeping Beauty transposon system, as shown in (Figure 4C). The results showed dose-dependent responses upon stimulation with different concentrations of melatonin. Therefore, we selected this composite part as the optimal component and constructed a cell-based screening platform based on P_4×CRE->IgK->Nluc->bGH polyA (BBa_K5267040) for our iGEM project.

reference

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