Difference between revisions of "Part:BBa K3734013"

We used NO21 miRNA (miR21), it can not only suppress the expression of target gene by targeting miR21T, but also speed the degradation of mRNA up.

Fig.1 The model diagram of pG-super-miR21

Fig.2 The model diagram of TRE-mCherry-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.

Fig.3 Electrophoretic diagram of miR21 PCR product

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

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.

[1]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.