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Revision as of 03:41, 25 September 2015
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
how you used this part and how it worked out.
how you used this part and how it worked out.
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<html>
===Applications of BBa_K1859016===
===Applications of BBa_K1859016===
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<blockquote>
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<br><br>
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We inserted this generator to <i>E.coli</i> and measure efficiency of circularization.<br>
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We extracted total RNA, reverse transcribed, conducted semi-quantitative PCR.<br>
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As a result, this generator circularized more efficiently than normal (no complementary sequence) one.[<a href= "http://2015.igem.org/Team:Gifu/result-page" style="text-decoration:none;" >Gifu 2015 RESULT</a>]<br><br>
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<b>Detecting circular mRNA</b><br>
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<img src="http://2015.igem.org/wiki/images/0/00/Gifu-rnase.png" width="300" height="293" vspace="25" hspace="50" align="right"></img>
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The existence of circular mRNA is confirmed by RNase processing. Endogenous RNA (linear RNA)(GAPDH) is decomposed by RNase R (exoribonuclease), but circular RNA is not decomposed. <br>
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Double-stranded DNA from undecomposed RNA can be gained with RT-PCR. This is how the existence of circular mRNA is confirmed by the observation of the DNA with electrophoresis.<br><br>
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<img src="http://2015.igem.org/wiki/images/c/c8/Gifu-pic2.png" width="322" height="152" vspace="10" hspace="30" align="right"></img>
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<br clear="all">
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<b>Fig. As a result of qualitative experiment of Circular mRNA</b></div>
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<font size="2"face="Arimo">
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<p>1:normal(iGEM Gifu 2014) <span> </span> 2:outside complementarity <span> </span> 3:inside complementarityⅠ<span> </span> 4:inside complementarityⅡ
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</p>
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<p>a…To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseR processing.</p>
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<p>b…To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseR processing.</p>
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<p>c…To detect the sequence of the circular mRNA in the cDNA derived from the non-treated RNA.</p>
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<p>d…To detect the sequence of the linear mRNA in the cDNA derived from the non-treated RNA.</p>
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<p>e…To detect the sequence of the circular mRNA in the non-treated RNA</p>
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<p>f…To detect the sequence of the linear mRNA in the non-treated RNA</p>
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<p>M…marker</p>
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</font>
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<blockquote>
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According to the picture (show above), this generator secceeded in making the circular mRNA.<br><br><br>
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<b>Semi-quantitative PCR</b>
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<br clear="all">
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<p>
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We introduced plasmid of "normal
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<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1332011" > [BBa_K1332011] </a>
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", "outside
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<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859026" > [BBa_K1859026] </a>
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", inside Ⅰ
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<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859024" > [BBa_K1859024] </a>
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, and inside Ⅱ
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<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859025" > [BBa_K1859025] </a>
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to <i>E. coli</i>, and pre-incubated each in 37℃ over night. Then we cultured with shaking the pre-culture liquids 100 µL with LB-Cm 10 mL in 37 for four hours and extracted total RNA from them with RNA extraction kit.
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After then, we reverse transcribed RNA by PCR and amplified certain region we targeted. One is a unique region of circular mRNA [ <b>region C</b> ], which includes joint region of circularization [ <b>region D</b> ]. The other is a common domain among circular and un-circular mRNA. We conducted reverse transcription PCR and PCR for the target. In the PCR, we performed 10 kinds of reaction which had the different number of cycles(12,14,16..30) for each sample. Then, we applied them in agarose gel and electrophoreses. And, we analyzed the data by using "imageJ". <br>
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<br>
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<img src="http://2015.igem.org/wiki/images/b/b6/Gifu-setumei.png" width="798" height="123" vspace="25" hspace="" align=""></img>
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</p></br>
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<p> The result of Semi-quantitative PCR was shown below. And, we made the graphs by using the raw data.
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<br>
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</p>
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<p>
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<img src="http://2015.igem.org/wiki/images/b/ba/OK1.JPG" width="240" height="180"></img>
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<img src="http://2015.igem.org/wiki/images/9/92/GelgelOK3.jpg" width="240" height="180"></img>
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<img src="http://2015.igem.org/wiki/images/5/5f/GelgelOK4.jpeg" width="240" height="180"></img>
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<br>
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<div class="center"><b>Fig. As a result of quantitative experiment of Circular mRNA </b></div>
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</p>
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<br>
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<p class="center"><b>Table.1 The mean of the fluorescence (tripartite)</b></p>
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<img src="http://2015.igem.org/wiki/images/c/ce/Hyou-gifu.png" width="790" height="173" vspace="10" hspace="40" align="right"></img>
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<br clear="all">
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<img src="http://2015.igem.org/wiki/images/f/fa/Gifu-fig-fig.png" width="817" height="125" vspace="10" hspace="40" align="right"></img>
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<br clear="all">
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<div class="center">
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<b>Fig. Relationship of fluorescence and cycle number</b>
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<p>From the left, normal, outside, insideⅠ and insideⅡ</p>
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</div>
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<br>
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<div class="center"><b>Table.2 Ct value at fluorescence 1.90</b><br>
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<img src="http://2015.igem.org/wiki/images/1/1b/Gifu-kekka-kekka.png" width="450" height="96" vspace="10" hspace="30"></img></div>
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<br clear="all">
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<p> According to this result, we calculated Ct value which indicates 1.90 fluorescence intensity. These value is summarized right. Ct is a value that the more the gene template in PCR increases, the more Ct value decreases. If there is a difference between “C” and “D”, there is a difference in the quantity of template geneThus, the difference between “C” and “D” is small; the cyclization may be efficiency.As a result, Ct in "
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normal <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_1332011" > [BBa_1332011]</a>
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" was 2.31, but “
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outside <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_1859026" > [BBa_1859026]</a>
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”, “
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inside Ⅰ <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_1859024" > [BBa_1859024]</a>
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”, “
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inside Ⅱ <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_1859025" > [BBa_1859025]</a>
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” were 1.28, 2.02, 1.89 collectively. From these things, it can be said that the efficiency of cyclization rose with all devices which we designed in this time. Especially, in the case of “outside”, there was a difference with Ct more than 1 point than “normal”. When we think that a quantity of the gene doubles for 1 cycle in PCR simply, it can be said that the cyclic efficiency of “outside” was twice as high as that of “normal”. <br>
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After all, it is thought that it fitted this splice site because it is a complementarity chain derived from the creature.
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</p>
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<br>
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<p class="center">
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<b>Table 3. Efficiency of circularization (relativity value)</b><br>
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<div class="">
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<table align="center" class="partsjump">
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<tr>
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<th></th><th>normal</th><th>outside</th><th>insideⅠ</th><th>insideⅡ</th>
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</tr>
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<tr>
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<td>Efficiency of circularization (relativity value)</td><td>1.00</td><td>2.05</td><td>1.22</td><td>1.34</td>
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</tr>
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</table>
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</div>
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</p>
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<br><br><br>
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</blockquote></blockquote>
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</body>
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</html>
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===User Reviews===
===User Reviews===