Difference between revisions of "Part:BBa K5267049"

Latest revision as of 02:55, 1 October 2024

P_min->IgK->Nluc->bGH_polyA

As a blank control

Sequence and Features

Profile

Name:Pmin>IgK->Nluc->bGH_polyA
Base pairs:859bp
Origin: Homo sapiens
Properties: As a blank control

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 dependent[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.

In part P_4xCRE>IgK->Nluc->bGH_polyA(Part:BBa_K5267040) We design a reporter plasmid vector for the melatonin receptor pathway .This part is designed to function as a control group. Pmin (Part:Bba_K3064030) is loaded into the vector as a diagnostic element to assess the successful activation of the receptor's downstream signaling pathways., IgK (Part:BBa_K3117006)is a signaling sequence, directing the protein into the secretory pathway , Nluc(c) 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 (Part: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. Howerer，since there is no response element upstream, the Gene expression should be low.

Special design

In order to verify the influence of CRE（cAMP response element） sequence on downstream pathway, we constructed Pmin using TATA box and GC box of CMW promoter. The Pmin is a low expression synthetic promoter in mammalian cells. CRE sequences can be placed upstream of this promoter to produce high expression of downstream genes

Function test

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[4], which can stimulate cAMP concentration to increase.</p>

To validate this part, initiating transcriptional activation, Weconstructed pNC005 pLM010, a plasmid carrying P_4xCRE-> IgK->Nluc->bGH_polyA (Part:BBa_K5267040) and Pmin->IgK->Nluc->bGH_polyA (Part:BBa_K5267049). When When transfected cells were stimulated by Forskolin causing intracellular cAMP concentration increased, the two groups are reciprocally used as control groups.

result

The result shows that a significant increase of the expression of Nluc gene compared to the 0xCRE control group, indicating that in the experimental group, the CRE sequence responded successfully. The result also successfully proved if there is no response element upstream, the Gene expression should be low.

HEK-293T cells were transfected with plasmids containing different promoters with 1×/5×/6×/7×NFAT elements respectively. Data are mean±SD of NanoLuc expression levels measured at 48 h after thapsigargin stimulation (n = 3 independent experiments).Upon a 48-hour incubation period, stimulation of the 1xNFAT promoter with 10 nM thapsigargin resulted in a mean augmentation of the NanoLuc reporter gene expression to a magnitude that was 1.96-fold superior to that ascertained in the absence of thapsigargin induction.

Reference

[1] H. E. Hamm, "How activated receptors couple to G proteins," Proc Natl Acad Sci U S A, vol. 98, no. 9, pp. 4819-21, Apr 24 2001, doi: 10.1073/pnas.011099798.

[2] D. Chen, J. Wang, J. Cao, and G. Zhu, "cAMP-PKA signaling pathway and anxiety: Where do we go next?," Cell Signal, vol. 122, p. 111311, Oct 2024, doi: 10.1016/j.cellsig.2024.111311.

[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.