Part:BBa_K3734022
TRE-mcherry-miR21
TRE is a DNA structure domain that combined with TetR. And miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene.
TRE-mCherry-miR21
TRE is a DNA structure domain that combined with TetR. It is a part of Tet-off system. This system consists of TetR and TRE can be blocked by tetracycline. Tetracycline will block the combination right between TetR and TRE and stop the system from working
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.
NO.21 miRNA (miR21) can not only suppress the expression of target gene by targeting miR21T, but also speed the degradation of mRNA up
1.Pattern diagram
Fig.1 The model diagram of TRE-mCherry-miR21
Fig.2 The model diagram of TRE-mCherry-miR21’s work
2.Experiment
2.1 Method
Phosphated ELK will activate the expression of downstream report gene mCherry after the combination of TRE and TetR. We used lase confocal microscope to observe the expression
As for miR21, we used 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
2.2 Result
TRE-mCherry-miR21 works well.
Fig.3 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
At the time of the design experiment, the first 48-hour test reported that the gene LUC/REN ratio was reported, and miR21 inhibited 40% of the expression, but this was not ideal for our design.
Fig.4 miR21 inhibited Luc expression 48h after transfection
Considering that it may be the reason for the continuous accumulation of LUC expression, we did another 24-hour group, and the results proved that miR21 suppressed 90% of the expression, and the experimental results were very in line with our design requirements.
Fig.5 miR21 inhibited Luc expression 24h after transfection
3.Caution
If the green fluorescent protein is expressed too much, and if the green fluorescence is too strong, it may cause a series of colors. In order to ensure the accuracy of the experimental results, the level of noise reduction should be raised.
The miR21 we built is a full-length sequence with flank sequences, which requires precursors such as pre and pri to be sheared and folded and matured within the cell. At the beginning of the experiment, we were worried about its inhibition efficiency and built miR21-stemloop, but because its efficiency can reach 90%, which fully meets our requirements, we still use the full length. Interested iGEMers can further test it.
Reference：
[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
[2]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.
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