Difference between revisions of "Part:BBa K5267005"

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==Special design==
 
==Special design==
 
<p> This basic part is a crucial element for testing whether the downstream pathway of melatonin responds successfully. Currently, a common method for studying signaling pathways involves cloning the response element of the transcription factor corresponding to the signaling pathway into a luciferase reporter gene vector, referred to as pCRE-luc.[2] However, the expression effect of a single response element is often weak; therefore, multiple tandem repeats of the same response element are typically inserted upstream of the reporter gene (in the 5'-UTR region) to enhance the activation of the signaling pathway[3]. By reviewing the literature, we constructed the 5xCRE-Pmin sequence, which contains a 5′ minimal promoter incorporating five multimerized palindromic CREs, potentially strengthening downstream gene expression. </p>
 
<p> This basic part is a crucial element for testing whether the downstream pathway of melatonin responds successfully. Currently, a common method for studying signaling pathways involves cloning the response element of the transcription factor corresponding to the signaling pathway into a luciferase reporter gene vector, referred to as pCRE-luc.[2] However, the expression effect of a single response element is often weak; therefore, multiple tandem repeats of the same response element are typically inserted upstream of the reporter gene (in the 5'-UTR region) to enhance the activation of the signaling pathway[3]. By reviewing the literature, we constructed the 5xCRE-Pmin sequence, which contains a 5′ minimal promoter incorporating five multimerized palindromic CREs, potentially strengthening downstream gene expression. </p>
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'''Figure 1.Schematic representation showing the construction of cAMP response element (CRE)-directed nanoluciferase(Nanoluc) reporter system.'''
  
 
==Function Test==
 
==Function Test==
 
===Method===
 
===Method===
 
<p> Forskolin (Coleonol) is a potent adenylate cyclase activator with an IC50 of 41 nM and an EC50 of 0.5 μM for type I adenylyl cyclase[4], which can stimulate an increase in cAMP concentration. </p>
 
<p> Forskolin (Coleonol) is a potent adenylate cyclase activator with an IC50 of 41 nM and an EC50 of 0.5 μM for type I adenylyl cyclase[4], which can stimulate an increase in cAMP concentration. </p>
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<p>To validate our basic part 5xCRE_Pmin (BBa_K5267005), which serves as the binding site for CREB and initiates transcriptional activation, we constructed the plasmid pNC005 pLM010. This plasmid carries 5xCRE_Pmin-IgK->Nluc->bGH_polyA (BBa_K5267041) and Pmin_IgK->Nluc->bGH_polyA (BBa_K5267049). When transfected cells were stimulated by Forskolin, theoretically, an increase in intracellular cAMP concentration should activate CREB, thereby binding to 5xCREs and initiating the transcription of this synthetic promoter 5xCRE_Pmin, and increase the expression of NanoLuc. </p>
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<p>To validate our basic part 5xCRE_Pmin ([https://parts.igem.org/Part:BBa_K5267005 Part:BBa K5267005]), which serves as the binding site for CREB and initiates transcriptional activation, we constructed the plasmid pNC005 pLM010. This plasmid carries 5xCRE_Pmin-IgK->Nluc->bGH_polyA ([https://parts.igem.org/Part:BBa_K5267041 Part:BBa K5267041]) and Pmin_IgK->Nluc->bGH_polyA ([https://parts.igem.org/Part:BBa_K5267049 Part:BBa K5267049]). When transfected cells were stimulated by Forskolin, theoretically, an increase in intracellular cAMP concentration should activate CREB, thereby binding to 5xCREs and initiating the transcription of this synthetic promoter 5xCRE_Pmin, and increase the expression of NanoLuc. </p>
 
===Result===
 
===Result===
  
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<p> '''Figure 1:  The expression of Nluc gene in different transfected cells was stimulated by forsklin for 48h.''' </p>
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<p> '''Figure 2:  The expression of Nluc gene in different transfected cells was stimulated by forsklin for 48h.''' </p>
 
<p> The result shows a significant increase of the expression of NanoLuc gene in 5xCRE_Pmin group upon the stimulation of forsklin compared to the control group, indicating that in the experimental group, the synthetic promoter 5xCRE_Pmin  sequence responded to increased cAMP concentration correctly and initiated transcription as expected.  </p>
 
<p> The result shows a significant increase of the expression of NanoLuc gene in 5xCRE_Pmin group upon the stimulation of forsklin compared to the control group, indicating that in the experimental group, the synthetic promoter 5xCRE_Pmin  sequence responded to increased cAMP concentration correctly and initiated transcription as expected.  </p>
  
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<p> '''Figure 2. Step-response dynamics of thansfeted cells under forsklin treatment. ''' </p>
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<p> '''Figure 3. Step-response dynamics of thansfeted cells under forsklin treatment. ''' </p>
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<p> Cell line carrying both PCRE5-Nluc and PCMV-MTNR1A expressing cassette was stimulated with the indicated amount of fosiklin 16 hours post seeding. Nanoluc expression levels were measured at different time points after melatonin stimulation (Data are mean±SD, n = 3 independent experiments). </p>
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<p> HEK293 cell line was co-transfected with both PCRE5-Nluc and PCMV-MTNR1A expressing cassette. Then the cell was exposed to a gradient concentration of forskolin to induce. NanoLuc expression levels were subsequently measured at different time points after forskolin stimulation. The results showed that cell could produce detectable amount of NanoLuc in a dose-dependent manner (Data are mean±SD, n = 3 independent experiments) as expected. These findings demonstrated that the effectiveness of cAMP pathway-responsive synthetic promoter. </p>
  
 
==Reference==
 
==Reference==

Latest revision as of 13:09, 2 October 2024


P_5xCRE

P_5xCRE is a synthetic promoter designed to respond to the activation of the cAMP/PKA/CREB pathway in mammalian cells.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 103
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Name: P_5xCRE
Base Pairs: 138bp
Origin: Homo sapiens
Properties: A synthetic promoter that responds to the activation of the cAMP/PKA/CREB pathway in mammalian cells.


Usage and Biology

The cAMP response element (CRE) plays a critical role as the binding site for the cAMP response element-binding protein (CREB), which is typically located within 100 nucleotides of the TATA box. CREB binds to cAMP response elements and recruits transcriptional coactivators, such as CBP/p300, to form transcription complexes that initiate the transcription of target genes.[1]

The TATA box is a fundamental component of eukaryotic promoters, typically represented by the consensus sequence TATA(A/T)A(A/T), located approximately 30 bp upstream of the transcription start site of most eukaryotic genes. This region is primarily composed of A-T base pairs and is essential for the selection of gene transcription, serving as a binding site for RNA polymerase, which must bind firmly to the TATA box to initiate transcription.

The ability of CRE sequences to mediate transcriptional activation in response to cAMP appears to be somewhat promoter-dependent[1].In this experiment, the TATA box of the commonly used CMV promoter was minimized to reduce the nucleotide sequence required for transcription, resulting in the designation of Pmin

The activity of CREB is modulated by various signaling cascades, including three downstream pathways activated by the melatonin receptors MT1 and MT2 in response to melatonin stimulation: the cAMP/PKA pathway, the calcium (Ca²⁺) signaling pathway, and the MAPK/ERK pathway. Consequently, CRE can be employed as a diagnostic element to assess the successful activation of the melatonin receptor's downstream signaling pathways.

Special design

This basic part is a crucial element for testing whether the downstream pathway of melatonin responds successfully. Currently, a common method for studying signaling pathways involves cloning the response element of the transcription factor corresponding to the signaling pathway into a luciferase reporter gene vector, referred to as pCRE-luc.[2] However, the expression effect of a single response element is often weak; therefore, multiple tandem repeats of the same response element are typically inserted upstream of the reporter gene (in the 5'-UTR region) to enhance the activation of the signaling pathway[3]. By reviewing the literature, we constructed the 5xCRE-Pmin sequence, which contains a 5′ minimal promoter incorporating five multimerized palindromic CREs, potentially strengthening downstream gene expression.

Figure 1.Schematic representation showing the construction of cAMP response element (CRE)-directed nanoluciferase(Nanoluc) reporter system.

Function Test

Method

Forskolin (Coleonol) is a potent adenylate cyclase activator with an IC50 of 41 nM and an EC50 of 0.5 μM for type I adenylyl cyclase[4], which can stimulate an increase in cAMP concentration.

To validate our basic part 5xCRE_Pmin (Part:BBa K5267005), which serves as the binding site for CREB and initiates transcriptional activation, we constructed the plasmid pNC005 pLM010. This plasmid carries 5xCRE_Pmin-IgK->Nluc->bGH_polyA (Part:BBa K5267041) and Pmin_IgK->Nluc->bGH_polyA (Part:BBa K5267049). When transfected cells were stimulated by Forskolin, theoretically, an increase in intracellular cAMP concentration should activate CREB, thereby binding to 5xCREs and initiating the transcription of this synthetic promoter 5xCRE_Pmin, and increase the expression of NanoLuc.

Result

Figure 2: The expression of Nluc gene in different transfected cells was stimulated by forsklin for 48h.

The result shows a significant increase of the expression of NanoLuc gene in 5xCRE_Pmin group upon the stimulation of forsklin compared to the control group, indicating that in the experimental group, the synthetic promoter 5xCRE_Pmin sequence responded to increased cAMP concentration correctly and initiated transcription as expected.

Figure 3. Step-response dynamics of thansfeted cells under forsklin treatment.

HEK293 cell line was co-transfected with both PCRE5-Nluc and PCMV-MTNR1A expressing cassette. Then the cell was exposed to a gradient concentration of forskolin to induce. NanoLuc expression levels were subsequently measured at different time points after forskolin stimulation. The results showed that cell could produce detectable amount of NanoLuc in a dose-dependent manner (Data are mean±SD, n = 3 independent experiments) as expected. These findings demonstrated that the effectiveness of cAMP pathway-responsive synthetic promoter.

Reference

[1] M. Montminy, "Transcriptional regulation by cyclic AMP," Annu Rev Biochem, vol. 66, pp. 807-22, 1997, doi: 10.1146/annurev.biochem.66.1.807.

[2] 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.

[3] 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.

[4] J. D. Robbins, D. L. Boring, W. J. Tang, R. Shank, and K. B. Seamon, "Forskolin carbamates: binding and activation studies with type I adenylyl cyclase," J Med Chem, vol. 39, no. 14, pp. 2745-52, Jul 5 1996, doi: 10.1021/jm960191+.