Difference between revisions of "Part:BBa K3064005"

Latest revision as of 02:05, 22 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.

The picture below shows this part's structure.

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 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

To optimize the design of our glucose sensing module, we designed promoter with three ChoRE binding minP(the left of pic). To characterize the 3xGSP promoter activation, Glucose sensing promoters (3xGSP) were cloned into pGL3 vector to control the expression of firefly luciferase. Corresponding plasmids were co-transfected into HepG2 cell line with SV40-rluc internal control plasmid. As is shown in the right of the picture below,comparing to mini-Promoter and taking renilla as the internal reference, 3xGSP showed significant increase of luciferase signal. This result strong proves 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 6: / Line 6: @@
 ===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. conducted experience to figure out the specific blood glucose concentration which can activate this composite part.
+The picture below shows this part's structure.
+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 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===
+To optimize the design of our glucose sensing module,  we designed promoter with three ChoRE binding minP(the left of pic). To characterize the 3xGSP promoter activation,   Glucose sensing promoters (3xGSP) were cloned into pGL3 vector to control the expression of firefly luciferase. Corresponding plasmids were co-transfected into HepG2 cell line with SV40-rluc internal control plasmid.  As is shown in the right of the picture below,comparing to mini-Promoter and taking renilla as the internal reference, 3xGSP showed significant  increase of luciferase signal. This result strong proves that CHoRE can significantly improve minP promoter’s promoting intensity.
+https://static.igem.org/mediawiki/parts/e/e5/T--NUDT_CHINA--2019pic3gsp1.jpg
 ===References===