Difference between revisions of "Part:BBa K3734013"
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The miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene. | The miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene. | ||
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<img width="400px" src="https://2021.igem.org/wiki/images/1/13/T--CSU_CHINA--tupian20.png"></p> | <img width="400px" src="https://2021.igem.org/wiki/images/1/13/T--CSU_CHINA--tupian20.png"></p> | ||
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<p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.2 The model diagram of TRE-mCherry-miR21</p> | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.2 The model diagram of TRE-mCherry-miR21</p> | ||
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<p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.3 Electrophoretic diagram of miR21 PCR product</p> | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.3 Electrophoretic diagram of miR21 PCR product</p> | ||
<p>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. | <p>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. | ||
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<p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.4 miR21 inhibited Luc expression 48h after transfection</p> | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.4 miR21 inhibited Luc expression 48h after transfection</p> | ||
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our design requirements. | our design requirements. | ||
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| − | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.5 miR21 inhibited Luc expression | + | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.5 miR21 inhibited Luc expression 24h after transfection</p> |
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| + | ===Usage and Biology=== | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 19:54, 20 October 2021
miR21
The miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene.
miR21
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
Fig.1 The model diagram of pG-super-miR21
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
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
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.
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