Team:CSU CHINA/Engineering
1.Overview
We wanted to accomplish a auto or semi-auto therapy during the brainstorm and design stage. As a medical measure, we have to make sure its safety, sensitivity and practicability. Therefore, we divided the whole loop into AND part and NOT part.
As for AND part, under the two conditions of hyperglycemia and the exposure of blue light, the engineered cell will secrete insulin and reduce the concentration of blood sugar. For NOT pary. When the secretion of insulin has reached certain level, the concentration of blood glucose is reduced, the cell will be activated and secrete miR21 to suppress the expression of insulin in a feedback way, forming a close loop to prevent hypoglycemia.
After reading literature and discussion, we have designed 17 basic parts and 8 composite part to complete our engineering and they have all expressed successfully. Our favourite parts are basic parts, BBa_K3734001 and BBa_K3734002 along with a composite part BBa_K3734030.
Fig.1 The model diagram of 9XUAS-Insulin
| Name | Description | Type | Function |
|---|---|---|---|
| BBa_K3734001 | CHREBPpromoter | DNA | Glucose-induced promoter |
| BBa_K3734002 | GIP promoter | DNA | Glucose-induced promoter |
| BBa_K3734030 | TRE-mCherry-miR21 | Composite | It is a part of the Tet-Off system and can express miR21 to achieve feedback inhibition |
Fig2.The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose cultur.
Fig3.Changes of LUC expression over time after blue light irradiation with different glucose concentrations
2.Engineering Success
2.1 CHREBP promoter (BBa_K3734001)
The abnormal of blood glucose concentration is important in various diseases but existing glucose-induced promoters are relatively few. We hope that these two glucose-induced promoters can be helpful for other iGEM teams. CHREBP is only used as a part of lipid metabolism currently, we use it as a glucose-induced promoter and achieved ideal wet lab result in 293T cells.
2.1.1 Model diagram
Fig.1 The model diagram of CHREBP-LUC
2.1.2 Luciferase detection system
2.1.2.1 Method
We use LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells
2.1.2.2 Result
Fig.2 Electrophoretic diagram of CHREBP PCR product
CHERBP promoter is induced by glucose and the expression rises along with the raise of glucose concentration.
Fig.3 The expression level of inducible promoter CHREBP was respectively analyzed at 48h in 25mM, 5.6mm and 0mM glucose cultur.
To eliminate the effect of residual glucose in the medium during transmission, we have conducted experiments in a 72-hour group, the result fits our anticipations more.
Fig.4 The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose culture
2.2 GIP promoter (BBa_K3734002)
GIP promoter is anticipated in current literature that it can only be used in several certain types of cells like intestine cells, but we proved that it can function will in 293T cells which are usually used in labs.
2.2.1 Model diagram
Fig.5 The model diagram of CHREBP-LUC
2.2.2 Real-time LUC
2.2.2.1 Method
We use LUC as report gene, test its real-time luminescence value at 560nm wavelength with specific devices and detect the expression level under different glucose concentration.
2.2.2.2 Result
GIP promoter is induced by glucose and the expression level rises along with the waise of glucose concentration.
Fig.6 Changes of LUC expression over time after blue light irradiation with different glucose concentrations
2.3 TRE-mCherry-miR21 (BBa_K3734030)
TRE-mCherry-miR21 is able to express miR21 and inhibit the combination with miR21T to inhibit the expression of target gene and is regulated by insulin.
2.3.1 Model diagram
Fig.7 The model diagram of TRE-mCherry-miR21
2.3.2 qPCR
2.3.2.1 Method
Prove miR21 is regulated by insulin and can target miR21 to inhibit the expression of target gene through detecting mRNA of report gene LUC and miRNA.
2.3.2.1 Result
We have proved that insulin can induce the expression of miR21 and inhibit the target gene from expressing, it fits our anticipation.
Fig.8 miR21 binds to miR21T and inhibits LUC expression
Fig.9 Insulin regulates miR21’s expression
Reference
[1] Jian Meng, Ming Feng, Weibing Dong.Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose[J].Sci Rep. 2016 Mar 31;6:23944.（CHREBP）
[2] Anthony T. Cheung,Bama Dayanandan,Jamie T. LewisGlucose-Dependent Insulin Release from Genetically Engineered K Cells[J].Science.2000,290:1959-1962.