Difference between revisions of "Part:BBa K3734022"

 
 
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<partinfo>BBa_K3734022 short</partinfo>
 
<partinfo>BBa_K3734022 short</partinfo>
  
 
TRE is a DNA structure domain that combined with TetR. And miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene.
 
TRE is a DNA structure domain that combined with TetR. And miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene.
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<head>
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</head>
  
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<body>
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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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                TRE-mCherry-miR21
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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>TRE is a DNA structure domain that combined with TetR. It is a part of Tet-off system. This system consists of TetR and TRE can be blocked by tetracycline. Tetracycline will block the combination right between TetR and TRE and stop the
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                    system from working
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                </p>
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                <p>mCherry is a monomer red fluorescent protein. Experiments proved that when it is combined with exogenous protein on both C side and N side, the activity of fluorescent and the function of combined proteinare not effected and it can be
 +
                    used together with multi types of fluorescent proteins. Due to the phosphorylation pathway is relatively long, we have added mCherry at the bottom as report gene to prove that our pathway works and is regulated by insulin.
 +
                </p>
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                <p>NO.21 miRNA (miR21) can not only suppress the expression of target gene by targeting miR21T, but also speed the degradation of mRNA up
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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/1/13/T--CSU_CHINA--tupian20.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 TRE-mCherry-miR21</p>
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                <p style="text-align: center;">
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                    <img width="396px" height="634" src="https://2021.igem.org/wiki/images/c/c0/T--CSU_CHINA--tupian18.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 The model diagram of TRE-mCherry-miR21’s work</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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                2.Experiment
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            </h2>
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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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                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>Phosphated ELK will activate the expression of downstream report gene mCherry after the combination of TRE and TetR. We used lase confocal microscope to observe the expression</p>
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                <p>As for 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
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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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                2.2 Result
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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>TRE-mCherry-miR21 works well.</p>
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/5/54/T--CSU_CHINA--tupian14.png"></p>
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                <!--put image's url here-->
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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.3 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system</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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                <br>
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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.</p>
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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.5 miR21 inhibited Luc expression 24h after transfection</p>
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                3.Caution
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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>If the green fluorescent protein is expressed too much, and if the green fluorescence is too strong, it may cause a series of colors. In order to ensure the accuracy of the experimental results, the level of noise reduction should be raised.
 +
                </p>
 +
                <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
 +
                    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.</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]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
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                </p>
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                <p>[2]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.</p>
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            </div>
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</body>
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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===

Latest revision as of 13:38, 20 October 2021

TRE-mcherry-miR21

TRE is a DNA structure domain that combined with TetR. And miR21 is a kind of microRNA, which can combined with miR21 target and inhabit the expression of target gene.

TRE-mCherry-miR21

TRE is a DNA structure domain that combined with TetR. It is a part of Tet-off system. This system consists of TetR and TRE can be blocked by tetracycline. Tetracycline will block the combination right between TetR and TRE and stop the system from working

mCherry is a monomer red fluorescent protein. Experiments proved that when it is combined with exogenous protein on both C side and N side, the activity of fluorescent and the function of combined proteinare not effected and it can be used together with multi types of fluorescent proteins. Due to the phosphorylation pathway is relatively long, we have added mCherry at the bottom as report gene to prove that our pathway works and is regulated by insulin.

NO.21 miRNA (miR21) 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 TRE-mCherry-miR21

Fig.2 The model diagram of TRE-mCherry-miR21’s work

2.Experiment

2.1 Method

Phosphated ELK will activate the expression of downstream report gene mCherry after the combination of TRE and TetR. We used lase confocal microscope to observe the expression

As for 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

2.2 Result

TRE-mCherry-miR21 works well.


Fig.3 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system

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

If the green fluorescent protein is expressed too much, and if the green fluorescence is too strong, it may cause a series of colors. In order to ensure the accuracy of the experimental results, the level of noise reduction should be raised.

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]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.

[2]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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1079
    Illegal NotI site found at 1085
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1055
    Illegal XhoI site found at 1
    Illegal XhoI site found at 1739
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2819