Part:BBa_K3064012
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. In addition, we increase the number of ChoRE to realize that engineered gene will be activated by particular high blood glucose concentration instead of normal blood glucose concentration.
The picture below shows this part's structure.
Figure 1.The structure of 9xGSP.
In this composite part, we used one minP and nine ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-9XGSP to verify its ability.
Characterization
Materials
PGL3-9XGSP
PGL3-miniP
Hepg2 cell line
Dual Luciferase Reporter Gene Assay Kit from Beyotime company
pcdna3.1-9xgsp-gfp
Method
First we choose luciferase as the reporting system.We separately transfected plasmids PGL3-9XGSP and PGL3-minP into HepG2 cells. After culturing these cells with glucose-free culture, we created low glucose concentration environment for cells and then stimulated cells with 20 mM glucose concentration culture. After 48 hours, these cells were gathered and dissoved for luciferase expression test with Dual Luciferase Reporter Gene Assay Kit of Beyotime company.
Figure 2. Transfection and Test of 9xGSP using luciferase
In other experiment, we adopt GFP to report the expression of the improved promoter. At the very first beginning, we starved the HepG2 cells with DMEM for 2 hours before transfection begins.After transfection 12h, we starved the cells with glucose-free culture and stimulate with 20mM glucose concentration culture after 6 more hours. Glucose stimulation intensity was controlled at 20mM. Samples were tested after transfection at different times.But this time we choose GFP/mcherry as indicators to show the expression level.Photograph makes the results clearer.
Figure 3.Transfection and Test of 9xGSP using GFP
Result
We chooose luciferase and GFP to report the experiment result respectively.
When we use luciferase as reporting system.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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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