Difference between revisions of "Part:BBa K3734013"

 
 
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<partinfo>BBa_K3734013 short</partinfo>
 
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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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<h2 class="pageContent-main__title">
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                <!--put tile here, <h2>title</h2>, class="pageContent-main__title" means it is the main title-->
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                miR21
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            <div class="pageContent-main__textBox">
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                <p>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.
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                <!--put text here, <p>content</p>-->
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                1.Pattern diagram
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/6/6d/T--CSU_CHINA--tupian22.png"></p>
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                <!--put image's url here-->
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of pG-super-miR21</p>
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/1/13/T--CSU_CHINA--tupian20.png"></p>
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                <br>
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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>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.Experiment
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            </h2>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.1 Method
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            <div class="pageContent-main__textBox">
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                <p>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.
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                </p>
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                <!--put text here, <p>content</p>-->
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.2 Result
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/4/44/T--CSU_CHINA--tupian24.png"></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>
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                <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>
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/5/51/T--CSU_CHINA--tupian25.png"></p>
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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>
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                <p>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
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                    our design requirements.
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                </p>
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/6/6d/T--CSU_CHINA--tupian3.png"></p>
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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 24h after transfection</p>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                3.Caution
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            <div class="pageContent-main__textBox">
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                <p>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
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                    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.
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                </p>
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                Reference：
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                <p>[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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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===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.

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]