Part:BBa_K5267004

P_4xCRE

As the response element to report whether melatonin is accepted or not 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 88
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: 4xCRE-Pmin
Base Pairs: 117bp
Origin: Homo sapiens
Properties: As the response element to report whether melatonin is accepted or not

Usage and Biology

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]

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.

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.

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.

Method

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. 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.

Result

< img src="https://static.igem.wiki/teams/5267/mao-parts/4xcre.jpg" class="figure-img img-fluid rounded" height="400px">

Figure 1: The expression of Nluc gene in different transfected cells was stimulated by forsklin for 48h 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. 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.

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] 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.
[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.
[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.
[5] 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+.