Difference between revisions of "Part:BBa K3064011"

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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 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.
+
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.  
  
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In this composite part, we used six ChoRE and one minP to engineer new promoter and conducted experience to figure out the specific blood glucose concentration which can activate this composite part.
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In this composite part, we used one minP and six ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-6XGSP to verify its ability.
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===Materials===
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PGL3-6XGSP
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PGL3-minP
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Hepg2 cell line
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Dual Luciferase Reporter Gene Assay Kit from  from Beyotime company
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===Method===
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We separately transfected plasmids PGL3-6XGSP and into hepg2 cells. After 48 hours, these cells were gathered and dissoved for luciferase expression test with Dual Luciferase Reporter Gene Assay Kit of Beyotime company.
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===Result===
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Taking renilla as the internal reference and comparing with the control experiment, it’s obvious to find that CHoRE can significantly improve minP promoter’s promoting intensity.
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https://static.igem.org/mediawiki/parts/b/bb/T--NUDT_CHINA--2019pic6xgsp.png
  
 
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Revision as of 23:29, 20 October 2019


6XGlucose Sensing Promoter

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

Usage and Biology

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.

In this composite part, we used one minP and six ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-6XGSP to verify its ability.

Materials

PGL3-6XGSP

PGL3-minP

Hepg2 cell line

Dual Luciferase Reporter Gene Assay Kit from from Beyotime company

Method

We separately transfected plasmids PGL3-6XGSP and into hepg2 cells. After 48 hours, these cells were gathered and dissoved for luciferase expression test with Dual Luciferase Reporter Gene Assay Kit of Beyotime company.

Result

Taking renilla as the internal reference and comparing with the control experiment, it’s obvious to find that CHoRE can significantly improve minP promoter’s promoting intensity. T--NUDT_CHINA--2019pic6xgsp.png

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]