Team:CSU CHINA/Best Composite Part
GIP-miR21T-GI-GAL4-4XmiR21T
1.Overview
GIP-miR21T-GI-GAL4-4XmiR21T is consists of glucose-induced promoter GIP, photosensitive protein GI, two yeast hybrid system part GAL4 and the target site of miR21: miR21T. It is regulated by both glucose concentration and blue light , enhanced the safety of this whole system. In the mean time, it is controlled by miR21 in a feedback way and form a close loop for the system, make it more sensitive for the reaction.
Only relying on the regulation by blood glucose may not be safe enough.Thus, we designed GI (BBa_K3734004)and LOV (BBa_K3734006) as a pair of photosensitive protein. Under the exposure of blue light, the construction of these 2 proteins will alter and have a mutual effect, forming a GAL4-GI-LOV-VP16 quadruple. GAL4 will recognize and combine with 9XUAS, makes it possible for VP16 to activate downstream expression.
2.Pattern diagram
Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T
3.Experiment
3.1 Method
We used report gene LUC to represent the effect of GIP-miR21T-GI-GAL4-4XmiR21T
3.2 Result
Fig.2 Light controlled system testing experiment 20210619
Fig.3 Expression level analysis under blue light irradiation and dark treatment
Fig.4 Instrument of shining blue light (450nm)
To test the status of our composite part, we improved the monitor method. We used real-time LUC as detection, got a better result
Fig.5 Changes of LUC expression over time after blue light irradiation with different glucose concentrations
4.Caution
(1)The duration of the combination between GI and LOV after the exposure of 450nm blue light will differ as the method and the intensity change. We used strong light for a 30min exposure and weak light for another 30min exposure in our experiment.
(2)GAL4 domain is more universal among eukaryotic cells, but the transcription activation factors cooperating with GAL4 like VP16 needs to be chosen differently according to different chassis creature.
(3)Use medium without glucose when paving in case the effect caused by residue glucose.
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.