Difference between revisions of "Part:BBa K3064012"

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9XGlucose Sensing Promoter

This composite part is a kind of improved promoter which is sensitive to particular high blood glucose concentration.

Usage and Biology

Hyperglycemia is a common symptom in Type two diabetic mellitus (T2D). In order to design a gene circuit that could ease symptoms of T2D automatically, sensing the high concentration of of blood glucose would be a essential and initial step of our degradation system. Thus, we design a new functional part that could respond to hyperglycemia and activate transciption of our degradation system based on an existing Part(BBa_M50098). Technically,a major glucose responsive transcription factor -- ChREBP would be dephosphorylated under high blood glucose. the dephosphorylated ChREBP would subsequently enter the nucleus to activate the gene expression of genes containing Carbohydrate-responsive element (ChoRE) sequence¹.Therefore, we design a novel promoter contains several ChREBP binding sites and a basic mini promoter.To enable robust ChREBP binding among different species, we integrated previously reported ChREBP ChIP-Seq data in both human and mouse to obtain reserved binding motif. Motif enrichment analysis provided us a minimum sequence of CHREBP binding site. Hence, we reasoned that a glucose sensitive transcriptional activation can be achieved by repeating such binding motif several times upstream of the minimum promoter. This part were designed to respond to glucose concentration by repeating ChoRE sqeuence nine times upstream of mini promoter, termed as 9X glucose-sensing promoter(9X GSP).
The figure below shows the structure of this glucose-sensing part.

Figure 1.The design and structure of 9X GSP.

Characterization

Materials

PGL3-9X GSP

PGL3-miniP

HepG2 cell line

Dual Luciferase Reporter Gene Assay Kit from Beyotime company

pcDNA3.1-9X GSP-gfp

Method

First, luciferase were chosen as the reporting system to quantitatively characterize the transcriptional strength of 9X GSP.
HepG2 cells were transfected with PGL3-9X GSP and PGL3-minP, separately. To validate the glucose responsiveness, we challenged the 9x GSP-luciferase carrying HepG2 cells by culturing cells in different concentration of glucose after overnight starvation. After 48 hours, cells were harvested and Dual Luciferase Reporter Gene assay were performed to measure the level of firefly luciferase transcribed by 9X GSP.

Figure 2. Characterization of 9X GSP using firefly luciferase

To further characterize the glucose sensing module, we generated a 9X GSP-GFP reporter plasmid to increase the detection throughput. By using CMV-mcherry to normalize the effect of glucose on general exogenous gene expression level. Specifically, HepG2 cells were starved overnight with glucose-free culture and stimulated with 20mM glucose concentration for 6 or more hours. Flurosecent images were subsequently obtained and analyzed by ImageJ softwar.

Figure 3.Characterization of 9X GSP using GFP

Result

Luciferase and GFP were independently utilized as the reporter gene to characterize 9X GSP.
In luciferase-based characterization, CMV-mcherry to normalize the effect of glucose on general exogenous gene expression level, we demonstrated that the 9x GSP is capable of activating gene expression in a glucose dose dependent manner. In the histogram below, the left is the result of PGL3-minP and the right is of PGL3-9XGSP. Taking renilla as the internal reference and comparing with the control experiment, we can find that the luciferase expression of PGL3-9XGSP is much higher than the expression of PGL3-minP. This result strong proves that CHoRE can significantly improve minP promoter’s promoting intensity.

Figure 4. The design and results of 9xGSP validation using luciferase.

When we use GFP as reporting system.After 18 hours’ transfection, we conduct experiments to test the function of our part. Photograph of fluorescence microscopy helps make results clear and obvious. Meanwhile, with the set of internal control, we can gain relative fluorescence intensity by Image J. During this process, we set different groups with different glucose concentration, which helps us to detect the relationship between GFP/mcherry and glucose concentration. Besides, test at different times makes the tendency of expression level as time passes much more clearer. From the figure we can easily discover that the glucose-sensing promoter can sense the glucose concentration and thus modify the expression level according to it. The figure below shows the results.

Figure 5. GFP/mcherry after 6 hours’ transfection(A). GFP/mcherry after 18 hours’ transfection(B). GFP/mcherry after 30 hours’ transfection(C). GFP/mcherry after 42 hours’ transfection(D). GFP/mcherry after 54 hours’ transfection(E).

References

[1] Li Ma,PengFei Gao,JianZhong Shi,et al.Research progress of ChREBP[J].Animal Husbandry and Feed Science,2014,35(09):40-42(Chinese)

Sequence and Features