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>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.
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</p>
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<div class="fig fig800">
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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>fig1. you have to change anti-sense RNA conforming with each target gene.</div>
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</div>
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<p></p><p></p>
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<p>We have a good idea! It is useful to use common anti-sense for different target gene.
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</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.
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</p>
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<p></p>
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<p>
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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>
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<p>
 +
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!!
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</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>
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<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li>
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<li>To control turbidity up to 0.1 at OD600</li>
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<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL)</li>
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<li>To measure fluorescence after 9 hour</li>
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</ol>
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<div class="fig fig800">
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<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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{{Team:HokkaidoU_Japan/JS}}

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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