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>How to synthesize antisense RNA
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<h1>How to synthesize anti-sense constructs
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<h4><p>Antisense-RBS fragments were synthesized based on BioBrick by primer annealing. The sides of antisense fragment have scar and restriction enzymes 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 antisense 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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<div class="fig fig400 para"><p>
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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-antisense 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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<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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<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 antisense worked. The colonies transformed antisense 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. Antisense 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