Difference between revisions of "Part:BBa K3064026"

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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 transcription. Therefore, we choose CHoRE as promoter of engineered plasmid and connect it with miniP 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/>
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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. <Br/>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Characterization===
 
===Characterization===
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This part BBa_K3064026 was cloned in pcDNA3.1+ and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page.Click https://2019.igem.org/wiki/images/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf to see more.<Br/>
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This part BBa_K3064026 was cloned in pcDNA3.1+ plasmid and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page.Click https://2019.igem.org/wiki/images/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf to see detail information.<Br/>
 
To conduct the later function test, we set two different groups to conduct the transfection. One is pcDNA3.1-9xGSP-GFP and the other one is plv-mcherry as the internal control. We transfected 300μg into HepG2 cells, which were cultured on 24-hole plate. When 90 percent  were mixed, we begin the transfection.  
 
To conduct the later function test, we set two different groups to conduct the transfection. One is pcDNA3.1-9xGSP-GFP and the other one is plv-mcherry as the internal control. We transfected 300μg into HepG2 cells, which were cultured on 24-hole plate. When 90 percent  were mixed, we begin the transfection.  
 
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 of 48h.<Br/>
 
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 of 48h.<Br/>
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==Special Design==
 
==Special Design==
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In order to improve this part, this year we have made a series of modification based on the Minimum TATA-box promoter designed by Daniel Tang of Stanford BIOE44 - S11.(BBa_M50098). Due to the low efficiency of TATA box promoter, we shorten the sequence into only minp. In addition, we also added glucose-sensing fragment to enhance the part’s initiation strength, as well as glucose-sensing function. With GFP, the part’s function can be better detected.
+
This year we have made a series of modification and functional improvements based on the Minimum TATA-box promoter designed by Daniel Tang of Stanford BIOE44 - S11.(BBa_M50098). In addition, we also added multiple glucose-sensing fragments to enhance the part’s activation strength under the high glucose concentration.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, named as 9X GSP.
  
 
https://2019.igem.org/wiki/images/a/ab/T--NUDT_CHINA--GSP_information.png
 
https://2019.igem.org/wiki/images/a/ab/T--NUDT_CHINA--GSP_information.png
 
Figure 2. The structure diagram of the 9xGSP-GFP part.
 
Figure 2. The structure diagram of the 9xGSP-GFP part.
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==Function Test==
 
==Function Test==
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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.
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Fluorescence protein-based characterization were performed to test the glucose-sensing function of promoter,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.  
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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.
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The figure below shows the characterization results.<Br/>
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https://2019.igem.org/wiki/images/e/eb/T--NUDT_CHINA--Test_of_function.png
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https://2019.igem.org/wiki/images/e/eb/T--NUDT_CHINA--Test_of_function.png<Br/>
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Figure 3. 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).
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Figure 3. 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.
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 +
==Reference==
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[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)

Latest revision as of 03:30, 22 October 2019


9xGSP-GFP

This composite part is made up of a kind of improved promoter which is sensitive to particular high blood glucose concentration and GFP sequence. The introduction of GFP makes it possible to examine the expression of this composite part. As designed,the part can be used as a glucose-sensing promoter with GFP reporter system.


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.
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]


Characterization

This part BBa_K3064026 was cloned in pcDNA3.1+ plasmid and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page.Click https://2019.igem.org/wiki/images/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf to see detail information.
To conduct the later function test, we set two different groups to conduct the transfection. One is pcDNA3.1-9xGSP-GFP and the other one is plv-mcherry as the internal control. We transfected 300μg into HepG2 cells, which were cultured on 24-hole plate. When 90 percent were mixed, we begin the transfection. 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 of 48h.
T--NUDT_CHINA--GSP_transfection_and_photograph.png
Figure 1. Steps of transfection and function test.

Special Design

This year we have made a series of modification and functional improvements based on the Minimum TATA-box promoter designed by Daniel Tang of Stanford BIOE44 - S11.(BBa_M50098). In addition, we also added multiple glucose-sensing fragments to enhance the part’s activation strength under the high glucose concentration.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, named as 9X GSP.

T--NUDT_CHINA--GSP_information.png Figure 2. The structure diagram of the 9xGSP-GFP part.

Function Test

Fluorescence protein-based characterization were performed to test the glucose-sensing function of promoter,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 3. 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.

Reference

[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)