Coding

Part:BBa_K3734013

Designed by: Xingjun Zhao   Group: iGEM21_CSU_CHINA   (2021-10-01)
Revision as of 10:25, 20 October 2021 by Shinichi (Talk | contribs)

miR21

The miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene. 

               miR21
           </h2>

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.

1.Pattern diagram

<img width="400px" src="T--CSU_CHINA--tupian22.png">

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

<img width="400px" src="T--CSU_CHINA--tupian20.png">

               

               <img width="400px" src="T--CSU_CHINA--tupian20.png">

Fig.2 The model diagram of TRE-mCherry-miR21

2.Experiment

2.1 Method

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

               <img class="image" src="T--CSU_CHINA--tupian24.png">

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. 

               <img class="image" src="T--CSU_CHINA--tupian25.png">

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. 

               <img class="image" src="T--CSU_CHINA--tupian3.png">

Fig.5 miR21 inhibited Luc expression 48h after transfection

3.Caution

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]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.

</body> </html> Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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