Team:HokkaidoU Japan/Projects/Length

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      Projects
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      Length Variation
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Introduction">Overview</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Introduction">Introduction</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
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<ul>
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
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            <li class="ldd_contents"><a href="">Overview</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Overview</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High School</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li>
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li>
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<p>
<p>
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It is known that the length of anti-sense is related to its repression efficiency (N Nakashima <i>et al.</i>, 2006<sup><a href="#cite-1">[1]</a></sup>), but the detail of relations between the length of anti-sense and their repression efficiency is still unclear. In this study, we made different lengths of anti-sense sequence (Fig. 1). </p>
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Using anti-sense RNA (asRNA) is one of the methods to repress gene expression.
 +
It is known that the length of anti-sense sequence is related to its repression efficiency (N. Nakashima <i>et al.</i>, 2006<sup><a href="#cite-1">[1]</a></sup>), but the details of the relation are still unclear. In this project, we made different lengths of anti-sense sequence (Fig. 1). </p>
<div class="fig fig800">
<div class="fig fig800">
<img src="https://static.igem.org/mediawiki/2014/8/8c/HokkaidoU_length_Overveiw2.png">
<img src="https://static.igem.org/mediawiki/2014/8/8c/HokkaidoU_length_Overveiw2.png">
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<div>Fig. 1 Each anti-sense repress mRNA.</div>
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<div>Fig. 1 Each anti-sense represses mRNA.</div>
</div>
</div>
<p>
<p>
-
Our experiments of anti-sense RNA (asRNA) sequence and its length reveals the relation. Thus our findings will be a clue for other iGEMers who wants to design their own anti-sense sequence.</p>
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Our experiments will be a clue for other iGEMers who want to design their own anti-sense sequence.</p>
<h1>Introduction</h1>
<h1>Introduction</h1>
<p>
<p>
-
In repressing gene by anti-sense RNA, it is important to determine the length of anti-sense. However, it is difficult to do it. Theoretically, if they are too long, it doesn’t repress target RNA effectively. The reason is RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them also gets low. However, too short asRNA also has some problem. The short anti-sense cannot bind to the specific part of mRNA because it has too short complementary sequences of target RNA. In the industrial and academic fields, people hope to use anti-sense that has suitable repression efficiency. For example, you can create knock down recombinant organisms easily by using strong anti-sense, and in iGEM, you can make bio-devices which have a complicated gene network and require fine-tuned gene expression. Gene expression is not only ON or OFF. As stated above, each cases need each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is found that this method is difficult.</p>
+
In repressing gene by anti-sense RNA, it is important to determine the length of anti-sense. However, it is difficult to do it. Theoretically, if the length is too long, it doesn’t repress target RNA effectively. The reason is because the RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them also gets low. However, too short asRNA also has some problems. The short anti-sense cannot bind to the specific part of mRNA because it has too short complementary sequences of target RNA. In the industrial and academic fields, people hope to use anti-sense that has suitable repression efficiency. For example, you can create knock down recombinant organisms easily by using strong anti-sense, and in iGEM, you can make bio-devices which have a complicated gene network and require fine-tuned gene expression. Gene expression is not only ON or OFF. As stated above, each case needs each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is known that this method is difficult.</p>
<div class="fig fig800">
<div class="fig fig800">
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<img src="https://static.igem.org/mediawiki/2014/b/bc/HokkaidoU_length_Length_gif.gif">
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<img src="https://static.igem.org/mediawiki/2014/8/81/HokkaidoU_length_Gifgifgifff.gif">
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<div>Fig. 2 Determining a repression region is defficult.</div>
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<div>Fig. 2 Determining a repression region is difficult.</div>
</div>
</div>
<p>
<p>
-
In this study, we made many kinds of anti-sense which have different lengths. These anti-sense constructs themselves is not useful for you. However, in our method, you can create the needed anti-sense. We expect this project will help you decide anti-sense sequences. We'd like to tell scientists and iGEMers how easy and acrate to repress by anti-sense RNA.</p>
+
In this project, we made many kinds of anti-sense which repress mRFP, but these anti-sense constructs themselves are not useful for you. However, in our method, you can create the anti-sense that you desire. We expect this project will help you decide anti-sense sequences. We’d like to tell scientists and iGEMers how easy and accurate to repress the gene expression by anti-sense RNA.</p>
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<h1 style="font-size:43px;" id="Method"> How to synthesize anti-sense constructs</h1>
+
<h1 style="font-size:43px;" id="Method">Methods</h1>
<p>
<p>
-
Inserted fragments were synthesized based on BioBrick by PCR. Forward primers are common (XhoI-Ptet (-10)). The primer binds to -10 region of Ptet (BBa_R0040) , and its end has XhoI restriction enzyme site. Each reverse primers are different (as90 NcoI, as120 NcoI) (Fig. 1). These primers bind to each specific part of mRFP (BBa_E1010) ,and their ends have NcoI restriction enzyme site. Then we got various length insert fragments, as90 and as120. As90 is the anti-sense that covers 90 bp of mRNA, and as 120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Of course, the sides of insert fragment have restriction enzymes XhoI, NcoI sites. </p>
+
We made anti-sense constructs that repress mRFP. The target construct are composed of P<sub>tet</sub> (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>), B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>), mRFP (<a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>) and double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).
 +
 
 +
 
 +
Insert fragments were synthesized based on BioBrick by PCR.  
 +
As forward primer, ”XhoI-P<sub>tet</sub> (-10)”was used for making all fragments. The primer binds to -10 region of P<sub>tet</sub>, and its end has XhoI restriction enzyme site. To change the downstream seqeunce, each reverse primer is designed differently (as90 NcoI, as120 NcoI) (Fig. 3). These primers bind to each specific part of mRFP, and their ends have NcoI restriction enzyme site. By that way, we can get various length of insert fragments, as90 and as120. As90 is the anti-sense that covers 90 bp of mRNA, and as120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Of course, the edges of insert fragments have restriction enzymes XhoI, NcoI sites.
 +
 
 +
</p>
<div class="fig fig800">
<div class="fig fig800">
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<img src="https://static.igem.org/mediawiki/2014/1/12/HokkaidoU_length_Length_method1.png">
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<img src="https://static.igem.org/mediawiki/2014/e/ee/HokkaidoU_length_Figfig3.png">
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<div>Fig. 3 Synthesizing anti-sense by PCR</div>
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<div>Fig. 3 Synthesizing insert fragments by PCR</div>
</div>
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<p>
<p>
-
<!
+
 
<p>
<p>
-
After we finished synthesizing insert fragments, we cut them and H-stem vector (our anti-sense expression vector) by XhoI and NcoI. Finally, we ligated them. The anti-sense constructs are complete because the insert fragments are ligated reversely. Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector and anti-sense B0034 examination. We performed repression examination by using their 4 anti-sense constructs.
+
After we finished synthesizing insert fragments, we inserted them into our H-stem vector (anti-sense expression vector <a href="http://parts.igem.org/Part:BBa_K1524100">BBa_K1524100</a>) by XhoI and NcoI.  
 +
 
 +
Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector in other experiment (as30 (<a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a>) and as60 (<a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem system</a>)). We performed repression experiment by using their four anti-sense constructs.
</p>
</p>
<div class="clearfix">
<div class="clearfix">
</div>
</div>
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<h1>How to assay</h1>
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<h2>How to assay</h2>
<p>
<p>
We performed RT-PCR to confirm the transcription of anti-sense RNA (asRNA) constructs.</p>
We performed RT-PCR to confirm the transcription of anti-sense RNA (asRNA) constructs.</p>
<ol>
<ol>
-
  <li>Cultivate the colony in 4 mL LB medium for 16 hours.</li>
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  <li>Cultivated the colony in 4 mL LB medium for 16 hours.</li>
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  <li>Centrifuge the 4 mL of culture at 10,000 rpm / for 2 min / at 25&deg;C</li>
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  <li>Centrifuged the 4 mL of culture at 10,000 rpm / for 2 min / at 25&deg;C</li>
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  <li>Remove the supernatant and add M9ZB medium then voltex the pelet.</li>
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  <li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li>
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  <li>Perform RT-PCR</li>
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  <li>Performed RT-PCR</li>
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  <li>Measure absorbance of 260nm about cDNA.</li>
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  <li>Measured absorbance of 260 nm about cDNA.</li>
</ol>
</ol>
-
<h1>Results</h1>
+
<h1 id="Results">Results</h1>
<p>
<p>
-
Though we measured absorbance of 260nm about cDNA, we could not get any cDNA. After RNA extraction, we confirmed absorbance of 260nm (this is absorbance of nucleic acid). However, after RT-PCR of that prodocuts, we could not confirm the existence of nucleic acid.  
+
Though we measured absorbance of 260 nm about cDNA, we could not get any cDNA. After RNA extraction, we confirmed absorbance of 260 nm (this is the absorbance of nucleic acid). However, after RT-PCR of that products, we could not confirm the existence of nucleic acid.
-
<br>Here, we show the discussion.</p>
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<p>
<p>
-
We estimated there is some problems in RNA extraction. First, we maybe loss RNA during the experiment operation. RNA is degraded easily than DNA because RNase is through the world, soil, air and water, of course in laboratories and human's bodies. We seemed to be careless for about it and overlook RNase's contamination.</p>
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It is presumable that there are some problems in RNA extraction. First, we might have lost RNA during the experiment operation. RNA is degraded easily than DNA because generally RNase can be easily contaminated.</p>
<p>
<p>
-
Second, Deactivation of DNase seemed incomplete. We used DNase at the end of RNA extraction because extracted sample contain DNA and RNA. To do it, we removed DNA and get only RNA. After all steps of RNA extraction, we deactivate DNase at 65&deg;C for 10 min. Probably, DNase was not deactivated. Because of it, DNase degraded cDNA produced in RT-PCR.</p>
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Secondly, the incomplete deactivation of DNase can be considered to be the reason of our failure. If DNase was not deactivated sufficiently, it will result in the degradation of cDNA produced in RT-PCR.</p>
<p>
<p>
-
Though we could not be in wiki freeze, we are going to retry. We will perform it being careful in these problems.</p>
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Though we could not make it by wiki freeze, we are going to retry this experiment.</p>
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<h1 id="Conclusion">Future Work</h1>
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<h1 id="Conclusion">Developmental experiment</h1>
<p>
<p>
-
We theoretically estimated the repression efficiency of anti-sense is related to the length. Therfore, we can make many kinds of repression efficiency anti-senses by making some length anti-sense. However, to synthesize many kinds of anti-senses, we must prepare each primers. As a future work, we propose an efficient method to synthesize various length of anti-sense.</p>
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We theoretically estimated that the repression efficiency of asRNA is related to its length. To find the optimum length, we must design many lengths of asRNA, and measure their efficiency. However, that takes a lot of time and labor. So, as a future work, we propose an efficient method to synthesize various length of anti-sense.</p>
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<h2>Method</h2>
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<h2>Methods</h2>
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+
-
<p>
+
-
Here, we explain this method by using mRFP expression construct as a target gene.</p>
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<h3>Preparation for randomizing</h3>
<h3>Preparation for randomizing</h3>
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-
 
-
 
-
 
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<div class="fig fig400" >
<div class="fig fig400" >
<img src="https://static.igem.org/mediawiki/2014/2/2c/HokkaidoU_length_Random1.png">
<img src="https://static.igem.org/mediawiki/2014/2/2c/HokkaidoU_length_Random1.png">
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<div>Fig.5 PrePCR for randomizing</div>
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<div>Fig. 5 PrePCR for randomizing</div>
</div>
</div>
<p>
<p>
<!
<!
<p>
<p>
-
Before randomizing, we have to perform some steps to make the effective anti-sense sequences .
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Before randomizing, we have to perform some steps to make the effective anti-sense fragments.
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First, we performed PCR on mRFP construct to use below primers.</p>
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First, we performed PCR on mRFP construct. We call this step "prePCR". We used below primers.</p>
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<p style="text-align:center;">
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<ul style="padding-left:50px; margin:20px 0;">
-
XhoI-pTet (-10)
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<li>XhoI-P<sub>tet</sub> (-10)</li>
 +
<li>mRFP 400 down</li>
 +
</ul>
 +
 
 +
XhoI-P<sub>tet</sub> (-10) is a primer that binds to -10 region of P<sub>tet</sub> (BBa_R0040), and its 5’contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). Because it doesn’t contain -35 region, DNA synthesizing starts from P<sub>tet</sub>’s -10 region and PCR products don’t contain a functional part as promoter.
<br>
<br>
-
mRFP 400dn</p>
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mRFP 400 down is a primer that binds to mRFP (BBa_E1010) 400 bp downstream.  
-
<p>
+
-
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;XhoI-pTet (-10) is a primer that binds to -10 sequence of Ptet (BBa_R0040), and its 3 ‘ contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). Because it doesn’t contain -35 sequence, DNA synthesizing starts from P<sub>tet</sub>’s -10 sequence and PCR products don’t contain a functional part as promoter.
+
<br>
<br>
-
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;mRFP 400dn is a primer that binds to mRFP (BBa_E1010)’s 400 downstream.
+
PCR products that are amplified by these 2 primes showed in Fig. 5.
-
<br>
+
-
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR products that are amplified by these 2 primes showed in Fig. 1.
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</p>
</p>
<div class="clearfix">
<div class="clearfix">
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<p>
<p>
-
Through this step, we can get about above 100bp fragments that seem the best length as effective anti-sense and contain SD sequence and start codon.</p>
+
Through this step, we can get insert fragments containing SD (Shine-Dargalno) sequence and start codon that are important to effictive repression.</p>
<h3>Randomizing</h3>
<h3>Randomizing</h3>
<p>
<p>
-
Next, for DNA synthesizing, we used PCR products synthesized previous step. The recipe showed below.</p>
+
For this method, we designed two primers shown below. The second primer is a "random primer" which can bind with any sequence on the template DNA. By using these primers, we can synthesize various lengths of anti-sense.
-
<div class="fig fig800">
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<ul style="padding-left:50px; margin:20px 0;">
-
<img src="https://static.igem.org/mediawiki/2014/5/5c/HokkaidoU_length_Random2.png">
+
<li>XhoI-P<sub>tet</sub> (-10)</li>
-
<div>Fig. 6 Randomizing recipe</div>
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<li>NcoI-NNNNNN</li>
 +
</ul>
 +
</p>
 +
 
 +
<p>
 +
However, the random primer only has 6 nt of region that anneals with the template DNA. Eventually, the Tm value of this primer gets extremely low. This causes some difficulties to PCR. As a solution, we have to take three unusual steps for PCR.
 +
<br>
 +
First, to single-strandize the template DNA, putting it at 95&deg;C / for 3 min and then putting it in 0&deg;C water. By this procedure, the template DNA will be kept denatured in low temperature.
 +
<br>Second, to anneal the primer and amplify the sequences in low temperature, we use "Klenow Fragment", a polymerase whose optimum temperature is 37&deg;C. In this step, we can not amplify fragments, but can make templates of various lengths. 
 +
<br>In the third step, we amplify the templates we mentioned above at 68&deg;C just like the usual PCR. </p>
 +
 
 +
<div class="fig fig400" >
 +
<img src="https://static.igem.org/mediawiki/2014/f/f5/HokkaidoU_length_Random3.png">
 +
<div>Fig. 6 How to make random anti-sense</div>
</div>
</div>
<p>
<p>
-
We added Klenow fragment, that is a DNA polymerase functioning on 37C, to the general PCR reaction system using KOD Plus NEO. We used 2 primers.
+
By these methods, you can get various lengths of asRNA of target gene. These fragments are all in one mixture of PCR products. Thus, by digesting, ligating the fragments into pHN1257 vector (published by Nakashima) and transforming them to cells, you can get various colonies with different lengths of anti-sense. The colony with the optimum length of anti-sense must be on the plate!</p>
-
</p>
+
 +
<h2>Results</h2>
 +
<p>
 +
We carried out this plan into practice. We used mRFP as the target gene. After prePCR, we performed PCR by following the steps we described above. The protocol of PCR is shown below.
 +
<div class="fig fig800">
 +
<img src="https://static.igem.org/mediawiki/2014/2/2c/HokkaidoU_length_Last_recipe.png">
 +
<div>Fig. 7 Randomizing protocol</div>
 +
</div>
 +
<div class="clearfix">
 +
</div>
-
<p style="text-align:center;">
 
-
XhoI-Ptet (-10)
 
-
<br>
 
-
NcoI-NNNNNN</p>
 
-
 
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<p>
 
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NcoI-NNNNNN has random site containing any nucleotides (A, T, C, G), and these random primers bind to random site  of template. Theirs 3 ‘ contains NcoI recognition site that is imperative to ligate with H-stem vector. We explain function of these enzymes and primers in any steps.
 
-
<br>
 
-
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In 37C step, Klenow fragment work. It synthesize DNA between XhoI-Ptet (-10) binding site and NcoI-NNNNNN binding sites that are random. DNA amplified in 3hrs are measurable length. This length is important for next step.</p>
 
<div class="fig fig400" >
<div class="fig fig400" >
-
<img src="https://static.igem.org/mediawiki/2014/f/f5/HokkaidoU_length_Random3.png">
+
<img src="https://static.igem.org/mediawiki/2014/4/4c/HokkaidoU_length_Protocols.png">
-
<div>Fig. 7 How to make random anti-sense</div>
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<div>Fig. 8 Results of randomizing</div>
</div>
</div>
<p>
<p>
<!
<!
<p>
<p>
-
Next, KOD Plus NEO starts general PCR system. In this reaction system, XhoI-Ptet (-10) and DNA fragment amplified by Klenow fragment work as primers. XhoI-Ptet (-10) bind to specific site of template DNA we hope, but another each primers bind to their specific random site because their sequences are different. Therefore we get some length PCR products.
+
We performed an experiment in two protocols, A and B. The annealing temperature and PCR cycles are different beteween A and B. We inserted them into vectors and transformed them to cells. We got some colonies in both plates! More colonies appeared in Protocol B than Protocol A. After that, we performed colony PCR. However, we could not get the result of colony PCR.
</p>
</p>
<div class="clearfix">
<div class="clearfix">
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<h2>Discussion</h2>
-
<h3>Detail</h3>
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We use Klenow fragment for reaction for NcoI-NNNNNN as primer. This primer is only about 6 mer that binds to DNA. Therefore in the reaction of KOD Plus NEO, it cannot anneal DNA because of high temperature. However, to use Klenow fragment and react slowly, DNA synthesizing that use NcoI-NNNNNN as primer becomes possible. By the way, Klenow fragment becomes deactivation through KOD Plus NEO’s reaction system.
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Getting this result that the colony PCR had failed, we stopped this project. However, recently, we realized that the stem sequences have difficuties in PCR (according to Mr. Nakashima). Therefore, we cannot conclude that this project have completely failed.</p>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We used this DNA fragment as insert. We ligated them with the anti-sense vector, performed transformation in a tube and spread to a plate. Some inserts contain XhoI site and NcoI site at each ends and the other contain NcoI site at both ends (Fig. 4). However, because after through dephosphorylation we ligated them, applied inserts were selected automatically in transformation. </p>
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<h3>Reference</h3>
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<img src="https://static.igem.org/mediawiki/2014/1/1f/HokkaidoU_length_Right_and_wrong.png">
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<div>Fig. 8 2 kinds of products are synthesized.</div>
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     <ol class="citation-list">
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       <li id="cite-1">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li>
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       <li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li>
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Latest revision as of 15:21, 9 September 2015

Projects
Length Variation

Overview

Using anti-sense RNA (asRNA) is one of the methods to repress gene expression. It is known that the length of anti-sense sequence is related to its repression efficiency (N. Nakashima et al., 2006[1]), but the details of the relation are still unclear. In this project, we made different lengths of anti-sense sequence (Fig. 1).

Fig. 1 Each anti-sense represses mRNA.

Our experiments will be a clue for other iGEMers who want to design their own anti-sense sequence.

Introduction

In repressing gene by anti-sense RNA, it is important to determine the length of anti-sense. However, it is difficult to do it. Theoretically, if the length is too long, it doesn’t repress target RNA effectively. The reason is because the RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them also gets low. However, too short asRNA also has some problems. The short anti-sense cannot bind to the specific part of mRNA because it has too short complementary sequences of target RNA. In the industrial and academic fields, people hope to use anti-sense that has suitable repression efficiency. For example, you can create knock down recombinant organisms easily by using strong anti-sense, and in iGEM, you can make bio-devices which have a complicated gene network and require fine-tuned gene expression. Gene expression is not only ON or OFF. As stated above, each case needs each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is known that this method is difficult.

Fig. 2 Determining a repression region is difficult.

In this project, we made many kinds of anti-sense which repress mRFP, but these anti-sense constructs themselves are not useful for you. However, in our method, you can create the anti-sense that you desire. We expect this project will help you decide anti-sense sequences. We’d like to tell scientists and iGEMers how easy and accurate to repress the gene expression by anti-sense RNA.

Methods

We made anti-sense constructs that repress mRFP. The target construct are composed of Ptet (BBa_R0040), B0034 (BBa_B0034), mRFP (BBa_E1010) and double terminator (BBa_B0015). Insert fragments were synthesized based on BioBrick by PCR. As forward primer, ”XhoI-Ptet (-10)”was used for making all fragments. The primer binds to -10 region of Ptet, and its end has XhoI restriction enzyme site. To change the downstream seqeunce, each reverse primer is designed differently (as90 NcoI, as120 NcoI) (Fig. 3). These primers bind to each specific part of mRFP, and their ends have NcoI restriction enzyme site. By that way, we can get various length of insert fragments, as90 and as120. As90 is the anti-sense that covers 90 bp of mRNA, and as120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Of course, the edges of insert fragments have restriction enzymes XhoI, NcoI sites.

Fig. 3 Synthesizing insert fragments by PCR
Fig. 4 Ligate the insert fragment with H-stem vector

After we finished synthesizing insert fragments, we inserted them into our H-stem vector (anti-sense expression vector BBa_K1524100) by XhoI and NcoI. Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector in other experiment (as30 (Anti-sense B0034) and as60 (H-stem system)). We performed repression experiment by using their four anti-sense constructs.

How to assay

We performed RT-PCR to confirm the transcription of anti-sense RNA (asRNA) constructs.

  1. Cultivated the colony in 4 mL LB medium for 16 hours.
  2. Centrifuged the 4 mL of culture at 10,000 rpm / for 2 min / at 25°C
  3. Removed the supernatant and add M9ZB medium then voltex the pelet.
  4. Performed RT-PCR
  5. Measured absorbance of 260 nm about cDNA.

Results

Though we measured absorbance of 260 nm about cDNA, we could not get any cDNA. After RNA extraction, we confirmed absorbance of 260 nm (this is the absorbance of nucleic acid). However, after RT-PCR of that products, we could not confirm the existence of nucleic acid.

It is presumable that there are some problems in RNA extraction. First, we might have lost RNA during the experiment operation. RNA is degraded easily than DNA because generally RNase can be easily contaminated.

Secondly, the incomplete deactivation of DNase can be considered to be the reason of our failure. If DNase was not deactivated sufficiently, it will result in the degradation of cDNA produced in RT-PCR.

Though we could not make it by wiki freeze, we are going to retry this experiment.

Developmental experiment

We theoretically estimated that the repression efficiency of asRNA is related to its length. To find the optimum length, we must design many lengths of asRNA, and measure their efficiency. However, that takes a lot of time and labor. So, as a future work, we propose an efficient method to synthesize various length of anti-sense.

Methods

Preparation for randomizing

Fig. 5 PrePCR for randomizing

Before randomizing, we have to perform some steps to make the effective anti-sense fragments. First, we performed PCR on mRFP construct. We call this step "prePCR". We used below primers.

  • XhoI-Ptet (-10)
  • mRFP 400 down
XhoI-Ptet (-10) is a primer that binds to -10 region of Ptet (BBa_R0040), and its 5’contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). Because it doesn’t contain -35 region, DNA synthesizing starts from Ptet’s -10 region and PCR products don’t contain a functional part as promoter.
mRFP 400 down is a primer that binds to mRFP (BBa_E1010) 400 bp downstream.
PCR products that are amplified by these 2 primes showed in Fig. 5.

Through this step, we can get insert fragments containing SD (Shine-Dargalno) sequence and start codon that are important to effictive repression.

Randomizing

For this method, we designed two primers shown below. The second primer is a "random primer" which can bind with any sequence on the template DNA. By using these primers, we can synthesize various lengths of anti-sense.

  • XhoI-Ptet (-10)
  • NcoI-NNNNNN

However, the random primer only has 6 nt of region that anneals with the template DNA. Eventually, the Tm value of this primer gets extremely low. This causes some difficulties to PCR. As a solution, we have to take three unusual steps for PCR.
First, to single-strandize the template DNA, putting it at 95°C / for 3 min and then putting it in 0°C water. By this procedure, the template DNA will be kept denatured in low temperature.
Second, to anneal the primer and amplify the sequences in low temperature, we use "Klenow Fragment", a polymerase whose optimum temperature is 37°C. In this step, we can not amplify fragments, but can make templates of various lengths.
In the third step, we amplify the templates we mentioned above at 68°C just like the usual PCR.

Fig. 6 How to make random anti-sense

By these methods, you can get various lengths of asRNA of target gene. These fragments are all in one mixture of PCR products. Thus, by digesting, ligating the fragments into pHN1257 vector (published by Nakashima) and transforming them to cells, you can get various colonies with different lengths of anti-sense. The colony with the optimum length of anti-sense must be on the plate!

Results

We carried out this plan into practice. We used mRFP as the target gene. After prePCR, we performed PCR by following the steps we described above. The protocol of PCR is shown below.

Fig. 7 Randomizing protocol
Fig. 8 Results of randomizing

We performed an experiment in two protocols, A and B. The annealing temperature and PCR cycles are different beteween A and B. We inserted them into vectors and transformed them to cells. We got some colonies in both plates! More colonies appeared in Protocol B than Protocol A. After that, we performed colony PCR. However, we could not get the result of colony PCR.

Discussion

Getting this result that the colony PCR had failed, we stopped this project. However, recently, we realized that the stem sequences have difficuties in PCR (according to Mr. Nakashima). Therefore, we cannot conclude that this project have completely failed.

Reference

  1. N. Nakashima et al. (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Res 34: 20 e138