Team:HokkaidoU Japan/Projects/asB0034/Overview

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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-Stem System</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Overview">Anti-senseB0034</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Overview">Anti-sense B0034</a></li>
            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Overview">Overview</a></li>
            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Overview">Overview</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Method">Method</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Method">Method</a></li>
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<h1>Overview</h1>
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<p>Antisense RNA is studied actively over the world. However, reliable method for gene silencing has not been clear. It is hard to find efficient sequence of antisense RNA and to synthesize new antisense fragments every time you change target gene.  
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<p>Anti-sense RNA is studied actively over the world. However, reliable method for gene silencing has not been clear. It is hard to find efficient sequence of anti-sense RNA and to synthesize new anti-sense fragment in keeping with your target gene.  
</p>
</p>
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<div class="fig fig400">
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<div class="fig fig800">
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<img src="https://static.igem.org/mediawiki/2014/7/71/HokkaidoU_antisenseB0034_overview01.png">
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<img src="https://static.igem.org/mediawiki/2014/8/88/HokkaidoU_project_antisenseB0034_overview01_800.png">
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<div>caption</div>
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<div>fig1. you have to change anti-sense RNA conforming with each target gene.</div>
</div>
</div>
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<p></p><p></p>
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<p>Here we found that antisense-RBS(B0034) fragment, that is used by igemer commonly, work to silence several proteins. We synthesized antisense B0034 fragment, igemer often use that RBS. Regardless of target gene, only one antisense fragment works various proteins on common RBS, B0034.
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<p>We have a good idea! It is useful to use common anti-sense for different target gene.
</p>
</p>
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 +
<p></p><p></p>
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<div class="fig fig800">
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<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview02_800.png">
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<div>fig2. Image of common anti-sense RNA effects.</div>
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</div>
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 +
<p></p><p></p>
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<p>Here we found that anti-sense-RBS (B0034) fragment, which is used by many iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.
 +
</p>
 +
<p></p>
<p>
<p>
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We also synthesized antisense-B0032 fragment in order to achieve specific silencing gene. The combination of antisense fragment and RBS, that locates upstream of target gene makes change silencing protein.
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We also synthesized anti-sense-B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.
</p>
</p>
<p>
<p>
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Specific antisense-RBS fragment help you save labor to make new antisense RNA for each target genes. Fortunately, igem HokkaidoU team select tractable RBS for designing antisense-RBS fragments. You can use our antisense fragments without resynthesize constructs you had. You only have to do is add our antisense fragment to construct had target gene!!
+
Specific anti-sense-RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense-RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. What you only have to do is to add our anti-sense fragment to the construct with the target gene!!
</p>
</p>
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<div class="fig fig800">
 +
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png">
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<div>fig3. Anti-sense B0034 has specific effects to B0034, RBS.</div>
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</div>
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<h1 id="Method">How to synthesize anti-sense constructs</h1>
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<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p>
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<div class="fig fig400 para">
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<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png">
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<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div>
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</div>
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<div class="fig fig400 para"><p>
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<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png">
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<div>Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex. </div>
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</div>
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<div class="fig fig800">
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<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png">
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<div>Fig. 3 Blue; antisense B0034, B0032  Red; scar sequence  Green; NcoI site  Purple; XhoI site</div>
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</div>
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<div class="fig fig400 para">
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<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png">
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<div>Fig. 4 B0034 & B0032 sequence </div>
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</div>
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<div class="fig fig400 para">
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<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png">
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<div>Fig. 5 Our parts</div>
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</div>
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<div class="clearfix"></div>
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<h1><p>How to assay</p>
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<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p>
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 +
<ol>
 +
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li>
 +
<li>To control turbidity up to 0.1 at OD600</li>
 +
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL)</li>
 +
<li>To measure fluorescence after 9 hour</li>
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</ol>
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 +
<div class="fig fig800">
 +
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png">
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<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div>
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</div>
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</div>
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</div>
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Latest revision as of 09:43, 15 October 2014

Overview

Anti-sense RNA is studied actively over the world. However, reliable method for gene silencing has not been clear. It is hard to find efficient sequence of anti-sense RNA and to synthesize new anti-sense fragment in keeping with your target gene.

fig1. you have to change anti-sense RNA conforming with each target gene.

We have a good idea! It is useful to use common anti-sense for different target gene.

fig2. Image of common anti-sense RNA effects.

Here we found that anti-sense-RBS (B0034) fragment, which is used by many iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.

We also synthesized anti-sense-B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.

Specific anti-sense-RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense-RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. What you only have to do is to add our anti-sense fragment to the construct with the target gene!!

fig3. Anti-sense B0034 has specific effects to B0034, RBS.

How to synthesize anti-sense constructs

Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI.

Fig. 1 How to make anti-sense B0034 by primer annealing

Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex.
Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site
Fig. 4 B0034 & B0032 sequence
Fig. 5 Our parts

How to assay

We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.

  1. To cultivate the colony in 4 mL LB culture for about 20 hours
  2. To control turbidity up to 0.1 at OD600
  3. To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL)
  4. To measure fluorescence after 9 hour
Fig. 6 Anti-sense B0034 is induced by IPTG

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