Difference between revisions of "Part:BBa K3064012"

Revision as of 02:31, 21 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

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 nine ChoRE and apply luciferase gene as downstream reporter in plasmid PGL3-9XGSP to verify its ability.

Materials

PGL3-9XGSP

PGL3-minP

Hepg2 cell line

Dual Luciferase Reporter Gene Assay Kit from from Beyotime company

Method

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.

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.

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]

@@ Line 23: / Line 23: @@
 ===Method===
-We separately transfected plasmids PGL3-6XGSP 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.
+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.
 ===Result===