Difference between revisions of "Part:BBa K3064005"

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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.
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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.  
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    In this composite part, we used one minP and three ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-3XGSP to verify its ability.
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===Materials===
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PGL3-3XGSP
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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 PGL3-3XGSP 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/f/fa/T--NUDT_CHINA--2019pic3xgsp.png
  
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In this composite part, we used one minP and three ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-3XGSP. conducted experience to figure out the specific blood glucose concentration which can activate this composite part.
 
  
 
===References===
 
===References===

Revision as of 23:23, 20 October 2019

3XGlucose Sensing Promter

This composite part is a kind of promoter which is sensitive to particular 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 three ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-3XGSP to verify its ability.

Materials

PGL3-3XGSP PGL3-minP Hepg2 cell line Dual Luciferase Reporter Gene Assay Kit from from Beyotime company

Method

   We separately transfected plasmids PGL3-3XGSP 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--2019pic3xgsp.png


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]