Team:CSU CHINA/Best Parts Collection
1.Overview
In our design, the whole loop is divided into AND and NOT these 2 parts. The AND part is consists of three composite parts:GIP-miR21T-GI-GAL4-4XmiR21T, LOV-VP16 and 9XUAS-Ad-Insulin. These three composite parts can be regulated by glucose concentration and blue light exposure and is inhibited in a feedback way by miR21, makes it safer for our design to be a therapeutic method.
2.Experiments
2.1 Glucose induction
GIP promoter is a glucose induced promoter, the reasons why we think it can be regarded as the best basic part are as followed:
(1)GIP promoter used to be assumed that it can only be used in several certain types of cells like intestine cells, but we have proved that it can function well in 293T, a type of cell usually used in labs. We have increased its universality, this is a breakthrough we have made on current literature
(2)The abnormality of glucose concentration can be important to lots of diseases, as for synthetic biology, the regulation of blood sugar is meaningful. It has a wide range of applications. However, current regulators are mostly lactose or other substances and current glucose induced promoter is few. We think these 2 glucose induced promoter can provide help for future iGEM teams.
(3)We designed different glucose concentration gradient through real-time LUC and sifted the best concentration, laid a foundation for future iGEMers’ experiments.
2.1.1 Pattern diagram
Fig.1The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T
2.1.2 Real-time LUC
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
GIP promoter is induced by glucose and the expression rises along with the raise of glucose concentration.
Fig.3 Light controlled system testing experiment 20210619
Fig.4 Expression level analysis under blue light irradiation and dark treatment
Fig.5 Instrument of shining blue light (450nm)
2.3 Insulin secretion
Naturally, a mature insulin molecule should go through precursors like preproinsulin and proinsulin to be secreted and make a difference. This means our cells need to be able to:(1) process insulin; (2) secrete insulin. Based on these two demands, we chose 293T. Meanwhile, we used gene Insulin(BBa_K3734015) to replace the report gene LUC(BBa_K3734014) we mentioned above. During the experiment, we used the specific ELISA tool kit for human mature insulin to test the supertanant and conducted experiments by deciding into two groups: dark and light group. The experiment proves that cell 293T can secret mature insulin outsides and is controlled by blue light.
Fig.6 Standard curve of absorbance and insulin concentration
Fig.7 Insulin concentration in supernatant after blue light irradiation and dark treatment respectively
Fig.8 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively
Reference
[1]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.
[2]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
[3]Anthony T. Cheung,Bama Dayanandan,Jamie T. LewisGlucose-Dependent Insulin Release from Genetically Engineered K Cells[J].Science.2000,290:1959-1962.