|BBa_K364304||TRE-CMV||Endre K roly Krist f|
All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.
TRE is a DNA structure domain that combined with TetR. It is a part of Tet-off system. Our first purpose of using it is to make phosphorylated ELK1 to locate and activate the expression of downstream genes (miRNA). The second reason we used it is because of the Tet-off system consists of TetR and TRE can be blocked by tetracycline. Tetracycline will stop the combination between TetR and TRE, stop the system from working. This provides a mandatory brake system for our loop. We can make sure the security and prevent the hypoglycemia caused by abnormal feedback through exogenous tetracycline.
Fig.1 The model diagram of TRE's work
mCherry is a monomer red fluorescent protein. Experiments proved that when it is combined with exogenous protein on both C side and N side, the activity of fluorescent and the function of combined proteinare not effected and it can be used together with multi types of fluorescent proteins. Due to the phosphorylation pathway is relatively long, we have added mCherry at the bottom as report gene to prove that our pathway works and is regulated by insulin.
Fig.2 The model diagram of TRE-mCherry-miR21
Fig.3 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
T21 is called miR21T in our project, it is a DNA sequence that can be the target of miRNA21. We have enhanced the method used by iGEM17_Greece in 2017 and inserted a piece of T21 in target gene 5’UTR and 4 pieces of T21s in 3’UTR, significantly improved the efficiency of miR21 targeting T21 and suppressing the expression of target gene, meet our feedback suppression requirement in our loop.
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.
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.