Difference between revisions of "Part:BBa K3064012"

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==Usage and Biology==
 
==Usage and Biology==
  
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Because there is high blood glucose concentration in diabetics, in order to get improved gene which is sensitive to diabetics it’s convenient to employ promoter which reacts to high blood glucose. Transcription factor -- ChREBP -- can be effectively expressed with high blood glucose. Meanwhile, heterodimer which consists of ChREBP and Mlx can combine with gene promoter ChoRE to induce gene transcription¹.Therefore, we choose ChoRE as promoter of engineered plasmid and connect it with minP to indicates a transcription start 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. <Br/>
+
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). <Br/>
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The picture below shows this part's structure. <Br/>
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The figure below shows the structure of this glucose-sensing part. <Br/>
 
https://2019.igem.org/wiki/images/9/9d/T--NUDT_CHINA--9xGSP_modified.png<Br/>
 
https://2019.igem.org/wiki/images/9/9d/T--NUDT_CHINA--9xGSP_modified.png<Br/>
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Figure 1.The structure of 9xGSP.<Br/>
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Figure 1.The design and structure of 9X GSP.<Br/>
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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==
 
==Characterization==
 
===Materials===
 
===Materials===
  
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PGL3-9XGSP
+
PGL3-9X GSP
  
 
PGL3-miniP
 
PGL3-miniP
  
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Hepg2 cell line
+
HepG2 cell line
  
 
Dual Luciferase Reporter Gene Assay Kit from Beyotime company
 
Dual Luciferase Reporter Gene Assay Kit from Beyotime company
  
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pcdna3.1-9xgsp-gfp
+
pcDNA3.1-9X GSP-gfp
  
 
===Method===
 
===Method===
  
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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.<Br/>
+
First, luciferase were chosen as the reporting system to quantitatively characterize the transcriptional strength of 9X GSP.<Br/>
 +
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.<Br/>
 
https://static.igem.org/mediawiki/parts/a/a6/T--NUDT_CHINA--Transfection_and_Test_of_9xGSP.png<Br/>
 
https://static.igem.org/mediawiki/parts/a/a6/T--NUDT_CHINA--Transfection_and_Test_of_9xGSP.png<Br/>
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Figure 2. Transfection and Test of 9xGSP using luciferase<Br/>
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Figure 2. Characterization of 9X GSP using firefly luciferase<Br/>
  
  
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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.<Br/>
+
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.<Br/>
 
https://static.igem.org/mediawiki/parts/c/c7/T--NUDT_CHINA--GSP-GFP_transfection.png<Br/>  
 
https://static.igem.org/mediawiki/parts/c/c7/T--NUDT_CHINA--GSP-GFP_transfection.png<Br/>  
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Figure 3.Transfection and Test of 9xGSP using GFP<Br/>
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Figure 3.Characterization of 9X GSP using GFP <Br/>
  
 
===Result===
 
===Result===
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We chooose luciferase and GFP to report the experiment result respectively.
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Luciferase and GFP were independently utilized as the reporter gene to characterize 9X GSP.<Br/>
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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.<Br/>
+
In luciferase-based characterization, SV40 promoter-renilla luciferase were utilized to normalize the effect of glucose on general exogenous gene expression level. The results indicated that luciferase expression activiated by 9X GSP is significantly higher than the expression activited by mini promoter. This result strongly proved that CHoRE can significantly improve mini promoter's transciptional strength under the high glucose concentration (20mM).<Br/>
 
https://static.igem.org/mediawiki/parts/9/97/T--NUDT_CHINA--Design_and_results.png<Br/>
 
https://static.igem.org/mediawiki/parts/9/97/T--NUDT_CHINA--Design_and_results.png<Br/>
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Figure 4. The design and results of 9xGSP validation using luciferase.<Br/>
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Figure 4. The functional validation of 9X GSP based on luciferase.<Br/>
  
  
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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.
+
In fluorescence protein-based characterization,CMV promoter-mcherry were used as internal control to normalize the effect of glucose on general exogenous gene expression level. At different time (6h, 18h, 30h, 42h, 54h) after transfection, fluorescence images were obtained by fluorescence microscope. Thus, fluorescence intensity of GFP could directly indicate the expression level of GFP under different glucose concentrations, which stands for transcriptional strength of 9X GSP. Results showed that 9X GSP is capable of activating gene expression in a glucose dose-dependent manner. <Br/>
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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.<Br/>
+
The figure below shows the characterization results.<Br/>
 
https://2019.igem.org/wiki/images/e/eb/T--NUDT_CHINA--Test_of_function.png<Br/>
 
https://2019.igem.org/wiki/images/e/eb/T--NUDT_CHINA--Test_of_function.png<Br/>
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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).
+
Figure 5. 9X GSP activites GFP expression in a glucose dose-dependent manner. Fluorescence images were obtained by fluorescence microscope and fluorescence intensity of GFP or RFP(mcherry) was calculated via calculating the integral value of grayscale intensity in RGB chunnel respectively. the intensity of mcherry were used to normalize the GFP expression. (A)The GFP/mcherry value 6 hours after transfection. (B) The GFP/mcherry value 18 hours after transfection. (C) The GFP/mcherry value 30 hours after transfection.(D)The GFP/mcherry value 42 hours after transfection. (E)The GFP/mcherry value 54 hours after transfection.
  
 
==References==
 
==References==

Latest revision as of 02:57, 22 October 2019


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.
T--NUDT_CHINA--9xGSP_modified.png
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.
T--NUDT_CHINA--Transfection_and_Test_of_9xGSP.png
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.
T--NUDT_CHINA--GSP-GFP_transfection.png
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, SV40 promoter-renilla luciferase were utilized to normalize the effect of glucose on general exogenous gene expression level. The results indicated that luciferase expression activiated by 9X GSP is significantly higher than the expression activited by mini promoter. This result strongly proved that CHoRE can significantly improve mini promoter's transciptional strength under the high glucose concentration (20mM).
T--NUDT_CHINA--Design_and_results.png
Figure 4. The functional validation of 9X GSP based on luciferase.


In fluorescence protein-based characterization,CMV promoter-mcherry were used as internal control to normalize the effect of glucose on general exogenous gene expression level. At different time (6h, 18h, 30h, 42h, 54h) after transfection, fluorescence images were obtained by fluorescence microscope. Thus, fluorescence intensity of GFP could directly indicate the expression level of GFP under different glucose concentrations, which stands for transcriptional strength of 9X GSP. Results showed that 9X GSP is capable of activating gene expression in a glucose dose-dependent manner.
The figure below shows the characterization results.
T--NUDT_CHINA--Test_of_function.png
Figure 5. 9X GSP activites GFP expression in a glucose dose-dependent manner. Fluorescence images were obtained by fluorescence microscope and fluorescence intensity of GFP or RFP(mcherry) was calculated via calculating the integral value of grayscale intensity in RGB chunnel respectively. the intensity of mcherry were used to normalize the GFP expression. (A)The GFP/mcherry value 6 hours after transfection. (B) The GFP/mcherry value 18 hours after transfection. (C) The GFP/mcherry value 30 hours after transfection.(D)The GFP/mcherry value 42 hours after transfection. (E)The GFP/mcherry value 54 hours after transfection.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]