Difference between revisions of "Part:BBa K5267006"
 
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<partinfo>BBa_K5267006 short</partinfo>
 
<partinfo>BBa_K5267006 short</partinfo>
  
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As the response element to report whether melatonin is accepted or not
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P_6xCRE is a synthetic promoter designed to respond to the activation of the cAMP/PKA/CREB pathway in mammalian cells.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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<br>Base Pairs: 159bp
 
<br>Base Pairs: 159bp
 
<br>Origin: Homo sapiens
 
<br>Origin: Homo sapiens
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<br>Properties: As the response element to report whether melatonin is accepted or not
+
<br>Properties: A synthetic promoter that responds to the activation of the cAMP/PKA/CREB pathway in mammalian cells.
  
  
 
===Usage and Biology===
 
===Usage and Biology===
−
<p>CRE(cAMP response element)play an important role as the binding site of CREB(cAMP response element binding protein) ,which is typically found 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 transcription of target genes.[1]</p>
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<p>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]</p>
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<p>TATA Box is one of the components that constitute the promoter of eukaryotes. The consistent order is TATA(A/T)A(A/T) (non-template chain sequence). It is about -30bp (-25~-32bp) upstream of the transcription starting point of most eukaryotic genes, and is basically composed of A-T base pairs, which determines the selection of gene transcription and is one of the binding sites of RNA polymerase. RNA polymerase can only start transcription after firmly binding to the TATA Box. The ability of CRE sequences to mediate transcriptional activation in response to cAMP appears to be somewhat promoter dependen[1],in this experiment, TATA box of the commonly used CMV promoter was selected to minimize to the minimum amount of nucleotides for transcription and named Pmin. </p>
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<p>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.</p>
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<p>The activity of CREB is modulated by a plethora of signaling cascades, including three downstream pathways that are activated by the melatonin receptor MT1/2 in response to melatonin stimulation: the cAMP/PKA pathway, the calcium (Ca2+) signaling pathway, and MAPK/ERK pathway.[2] Consequently, CRE can be employed as a diagnostic element to assess the successful activation of the melatonin receptor's downstream signaling pathways.</p>
+
<p>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</p>
−
 
+
<p>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.</p>
  
 
===Special design===
 
===Special design===
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In part 4xCRE-Pmin (BBa_K5267004), we demonstrated that CRE can promote downstream gene transcription after binding to CREB. However, we cannot be sure that 4 multimerized CRE sequence incorporating a 5'minimal promoter is the best choice to promote expression. In order to select the best number of clones，in this parts ，we constructed the 6xCRE-Pmin sequence, containing a 5'minimal promoter with 6 multimerized CREs.
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<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 6xCRE-Pmin sequence, which contains a 5′ minimal promoter incorporating six multimerized palindromic CREs, potentially strengthening downstream gene expression. </p>
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<html>
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<figure class="figure">
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<div style="width=100%; height=auto; text-align: center">
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<img src="https://static.igem.wiki/teams/5267/mao-parts/456cre.png" class="figure-img img-fluid rounded"  height="200px">
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</div>
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</figure>
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</html>
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'''Figure 1.Schematic representation showing the construction of cAMP response element (CRE)-directed nanoluciferase(Nanoluc) reporter system.'''
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==Function Test==
 +
===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>To validate our basic part 6xCRE_Pmin ([https://parts.igem.org/Part:BBa_K5267006 Part:BBa K5267006]), which serves as the binding site for CREB and initiates transcriptional activation, we constructed the plasmid pNC005 pLM010. This plasmid carries 6xCRE_Pmin-IgK->Nluc->bGH_polyA ([https://parts.igem.org/Part:BBa_K5267042 Part:BBa K5267042]) 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 6xCREs and initiating the transcription of this synthetic promoter 6xCRE_Pmin, and increase the expression of NanoLuc. </p>
 +
===Result===
  
 +
<html>
 +
<figure class="figure">
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<div style="width=100%; height=auto; text-align: center">
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<img src="https://static.igem.wiki/teams/5267/mao-parts/4xcre.jpg" class="figure-img img-fluid rounded"  height="400px">
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</div>
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</figure>
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</html>
  
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===Function Test===
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<p> '''Figure 2:  The expression of Nluc gene in different transfected cells was stimulated by forsklin for 48h.''' </p>
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==Method==
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<p> The result shows a significant increase of the expression of NanoLuc gene in 6xCRE_Pmin group upon the stimulation of forsklin compared to the control group, indicating that in the experimental group, the synthetic promoter 6xCRE_Pmin  sequence responded to increased cAMP concentration correctly and initiated transcription as expected. </p>
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Forskolin (Coleonol) is a potent adenylate cyclase activator with an IC50 of 41nM and an EC50 of 0.5μM for type I adenylyl cyclase[5], which can stimulate cAMP concentration to increase.
+
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To validate our basic part 4xCRE_Pmin(BBa), which can work as the binding site ofCREB(cAMP response element binding protein), initiating transcriptional activation, Weconstructed pNC005 pLM010, a plasmid carrying 4xCRE_Pmin- IgK->Nluc->bGH_polyA (BBa) and Pmin _IgK->Nluc->bGH_polyA (BBa). When When transfected cells were stimulated by Forskolin causing intracellular cAMP concentration increased, the two groups are reciprocally used as control groups.
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==Result==
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<html>
 
<html>
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<figure class="figure">
 
<figure class="figure">
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<div style="width=100%;height=auto">
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<div style="width=100%; height=auto; text-align: center">
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< img src="https://static.igem.wiki/teams/5267/mao-parts/4xcre.jpg" class="figure-img img-fluid rounded"  height="400px">
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<img src="https://static.igem.wiki/teams/5267/i-m-zhangrenjie/013.jpg" class="figure-img img-fluid rounded"  height="400px">
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</div>
 
</figure>
 
</figure>
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</html>
 
</html>
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Figure 1:  The expression of Nluc gene in different transfected cells was stimulated by forsklin for 48h
 
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The result shows that  a significant increase of the expression of Nluc gene compared to the control group, indicating that in the experimental group, the CRE sequence responded successfully. 
 
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<p>The result successfully proved our system can work as we expected—when cAMP concentration increases, the CREB will bind with CRE sequence promoting expression of gene downstream.</p>
 
  
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===Reference===
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<p> '''Figure 3. Step-response dynamics of thansfeted cells under forsklin treatment. ''' </p>
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[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.
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<p> HEK293 cell line was co-transfected with both PCRE6-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>
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<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.
+
 
−
<br>[3] 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+.
+
==Reference==
 +
<p> [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. </p>
 +
<p> [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. </p>
 +
<p> [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. </p>
 +
<p> [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+. </p>

Latest revision as of 13:07, 2 October 2024


6xCRE-Pmin

P_6xCRE 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 118
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Name: 6xCRE-Pmin
Base Pairs: 159bp
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 6xCRE-Pmin sequence, which contains a 5′ minimal promoter incorporating six 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 6xCRE_Pmin (Part:BBa K5267006), which serves as the binding site for CREB and initiates transcriptional activation, we constructed the plasmid pNC005 pLM010. This plasmid carries 6xCRE_Pmin-IgK->Nluc->bGH_polyA (Part:BBa K5267042) 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 6xCREs and initiating the transcription of this synthetic promoter 6xCRE_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 6xCRE_Pmin group upon the stimulation of forsklin compared to the control group, indicating that in the experimental group, the synthetic promoter 6xCRE_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 PCRE6-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+.