Team:HokkaidoU Japan/Projects/asB0034/Method

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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>RNA constructs
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<h1>How to synthesize anti-sense constructs
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<h4><p>Anti-sense RBS fragments were synthesized based on BioBrick by primer annealing. The sides of antisense fragment have scar and restriction enzymes XhoI, NcoI. To be suitable BioBrick, we add scar sequence to anti-sense RNA. We finished synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by restriction enzyme XhoI, NcoI. </p>
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<h4><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>
<div class="fig fig400 para">
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<img src="https://static.igem.org/mediawiki/2014/0/02/HokkaidoU_antisenseB0034_overview07.png">
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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>Fig1. How to make anti-sense B0034 by primer annealing</div>
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<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div>
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<img src="https://static.igem.org/mediawiki/2014/6/63/HokkaidoU_antisenseB0034_overview08.png">
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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>Fig2. Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex. </div>
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<div>Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex. </div>
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<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png">
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png">
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<div>Fig3. Blue; antisense B0034, B0032  Red; scar sequence  Green; NcoI site  Purple; XhoI site</div>
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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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<img src="https://static.igem.org/mediawiki/2014/e/e3/HokkaidoU_antisenseB0034_overview12.png">
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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>Fig4. Our parts</div>
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<div>Fig. 4 B0034 & B0032 sequence </div>
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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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<h1><p>How to assay</p>
 
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<h4><p>we selected RFP and GFP for target genes. We used fluorophotometer to measure how anti-sense worked. The colonies transformed anti-sense RNA and target gene used to do assay.</p>
 
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<h1><p>How to assay</p>
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<h4><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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<p>(1)To cultivate the colony in 4 mL LB culture for about 20 hours</p>
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<ol>
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<p>(2)To control turbidity up to 0.1 at OD600</p>
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<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li>
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<p>(3)To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL )</p>
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<li>To control turbidity up to 0.1 at OD600</li>
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<p>(4)To measure fluorescence after 9 hour </p>
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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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<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_antisenseB0034_overview09.png">
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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>Fig4. Anti-sense B0034 is induced by IPTG</div>
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<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div>
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Latest revision as of 11:58, 14 October 2014

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