Difference between revisions of "Part:BBa K3734024"

 
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<partinfo>BBa_K3734024 short</partinfo>
 
<partinfo>BBa_K3734024 short</partinfo>
  
 
CHREBP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of LUC. Luciferin(LUC) is a protein that can activate fluorescein.We use it as reporter gene.
 
CHREBP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of LUC. Luciferin(LUC) is a protein that can activate fluorescein.We use it as reporter 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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                miR21T-LUC-4XmiR21T
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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                <p>To prove miR21 can target miE21T and suppress the expression of the target gene, we have designed miR21T-LUC-4XmiR21T
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                </p>
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                <!--put text here, <p>content</p>-->
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                1.Pattern diagram
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            </h2>
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            <div class="pageContent-main__textBox">
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/5/59/T--CSU_CHINA--tupian58.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 miR21T-LUC-4XmiR21T</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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            </h2>
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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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. In the mean time, we tested the level of mRNA
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                    expression through qPCR.</p>
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                <!--put text here, <p>content</p>-->
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.2 Result
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            </h2>
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            <div class="pageContent-main__textBox">
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/8/86/T--CSU_CHINA--tupian59.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.2 Electrophoretic diagram of miR21T-LUC-4XmiR21T 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.</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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                <!--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.3 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.</p>
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                <p>In the mean time, we have tested miR21 to speed up the degradation of mRNA through qPCR.</p>
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            </div>
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            <p style="text-align: center;">
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                <img width="400px" src="https://2021.igem.org/wiki/images/b/be/T--CSU_CHINA--tupian26.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.4 miR21 inhibited Luc expression 24h after transfection</p>
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            <p style="text-align: center;">
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                <img width="400px" src="https://2021.igem.org/wiki/images/f/f4/T--CSU_CHINA--tupian62.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.5 miR21 promoted the mRNA degradation efficiency of LUC</p>
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                3.Caution
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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                <p>Because we have repeated mE21T for 4 times at 3’UTR area, we should avoid the repeated pieces when designing the primer. Please pay attention to how many times does miR21T have repeated in PCR outcome. Meanwhile, there is similar parts
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                    between miR21T and padrome structure, which should be paid attention to during the design</p>
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                <!--class="pageContent-main__title" means it is the sub title-->
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                Reference：
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            </h2>
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                <!--all the content must included in this div-->
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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===

Revision as of 13:52, 20 October 2021

miR21T-LUC-4XmiR21T

CHREBP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of LUC. Luciferin(LUC) is a protein that can activate fluorescein.We use it as reporter gene.

miR21T-LUC-4XmiR21T

To prove miR21 can target miE21T and suppress the expression of the target gene, we have designed miR21T-LUC-4XmiR21T

1.Pattern diagram

Fig.1 The model diagram of miR21T-LUC-4XmiR21T

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. In the mean time, we tested the level of mRNA expression through qPCR.

2.2 Result

Fig.2 Electrophoretic diagram of miR21T-LUC-4XmiR21T 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.3 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.

In the mean time, we have tested miR21 to speed up the degradation of mRNA through qPCR.

Fig.4 miR21 inhibited Luc expression 24h after transfection

Fig.5 miR21 promoted the mRNA degradation efficiency of LUC

3.Caution

Because we have repeated mE21T for 4 times at 3’UTR area, we should avoid the repeated pieces when designing the primer. Please pay attention to how many times does miR21T have repeated in PCR outcome. Meanwhile, there is similar parts between miR21T and padrome structure, which should be paid attention to during the design

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 166
    Illegal NgoMIV site found at 1510
    Illegal NgoMIV site found at 1531
    Illegal AgeI site found at 1234
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1416