Difference between revisions of "Part:BBa K3064011"
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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. | 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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| + | The picture below shows this part's structure. | ||
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. | 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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===Method=== | ===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. | + | 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. |
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| + | After the experiment above, we cultured hepg2 cells which were transfected by plasmid PGL3–6XGSP with a series of glucose concentration: 0mM, 5mM, 10mM, 15mM, 20mM and 30mM. And the other steps were same as previous experiment. | ||
===Result=== | ===Result=== | ||
| − | + | To optimize the design of our glucose sensing module, we designed promoter with six ChoRE binding minP(the left of pic). To characterize the 6xGSP 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, 6xGSP showed significant increase of luciferase signal. This result strong proves that CHoRE can significantly improve minP promoter’s promoting intensity. | |
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| + | https://static.igem.org/mediawiki/parts/0/05/T--NUDT_CHINA--2019pic6xgsp1.jpg | ||
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| + | The consequence of the second experiments. | ||
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| + | To validate the glucose responsiveness, we challenged the 6xGSP-luciferase carrying cells by culturing cells in 0, 5, 10, 15, 20 and 30mM glucose for 48h. Results showed that the luciferase expression of PGL3-6XGSP varies with the concentration of glucose and the expresion with 15mM is obviously hiher than with 5mM. This result strong proves that 6xGSP promter's ability of promting can vary with glucose concentration and can strongly promte its downstream gene with 15mM. | ||
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| − | + | [[File:T--NUDT_CHINA--2019pic6xgsp2.jpg|250px|]] | |
===References=== | ===References=== | ||
Latest revision as of 17:20, 21 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.
The picture below shows this part's structure.
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 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.
After the experiment above, we cultured hepg2 cells which were transfected by plasmid PGL3–6XGSP with a series of glucose concentration: 0mM, 5mM, 10mM, 15mM, 20mM and 30mM. And the other steps were same as previous experiment.
Result
To optimize the design of our glucose sensing module, we designed promoter with six ChoRE binding minP(the left of pic). To characterize the 6xGSP 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, 6xGSP showed significant increase of luciferase signal. This result strong proves that CHoRE can significantly improve minP promoter’s promoting intensity.
The consequence of the second experiments.
To validate the glucose responsiveness, we challenged the 6xGSP-luciferase carrying cells by culturing cells in 0, 5, 10, 15, 20 and 30mM glucose for 48h. Results showed that the luciferase expression of PGL3-6XGSP varies with the concentration of glucose and the expresion with 15mM is obviously hiher than with 5mM. This result strong proves that 6xGSP promter's ability of promting can vary with glucose concentration and can strongly promte its downstream gene with 15mM.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]