Difference between revisions of "Part:BBa K3734025"

GIP-miR21T-GI-GAL4-4XmiR21T consists of glucose-induced promoter GIP, photosensitive protein GI, double yeast hybrid system components GAL4 and miR21T. It is regulated by both glucose concentration and blue
light, which improves the safety of the whole system. At the same time, it is also suppressed by miR21 feedback, which makes the whole system form a closed loop 
and makes the reaction more sensitive.

In order to increase the safety of patients' use, it may be difficult to achieve the expected goal of accurate regulation by relying on the regulation of blood glucose 
concentration alone, so we designed a pair of photosensitive proteins 
of GI ( BBa_K3734033) and LOV (BBa_K3734033). Under blue light irradiation, the Protein structure changes and interacts with each other to form a GAL4-GI-LOV-VP16 quadruple, while GAL4 ( BBa_K3734033) identifies and combines 9XUAS (BBa_K3734033), so that VP16 can activate the gene of downstream expression.

Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T

We used report gene LUC to represent the effect of GIP-miR21T-GI-GAL4-4XmiR21T

Fig.2 Light controlled system testing experiment

Fig.3 Expression level analysis under blue light irradiation and dark treatment

The duration of GI and LOV after 450nm blue light exposure will vary with the irradiation method, intensity, etc. In the experiment, we adopted the method of irradiating strong light for 30 minutes first, and then low light for 30 minutes. The GAL4 binding domain is universal in eukaryotic cells, but transcription activators (such as VP16) that cooperate with GAL4 need to be properly selected according to different chassis organisms.

[1]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.

[2]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.

[3]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.