Difference between revisions of "Team:Tianjin/Note/CFPS"

 
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<div id="main">         
 
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        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<!--------------------------------week1 start-------------------------------------------->
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<div id="Week1"></div>       
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
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<div class="entry-title" align="center" ><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Notes</a></div>
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<div class="entry-title" align="center" >Notes For CFPS system</div>
 
</header><!-- .entry-header -->
 
</header><!-- .entry-header -->
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         <h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week1(8/1/2016-8/7/2016)</a></h1>
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         <h1 class="entry-title">Week1(8/24/2016-8/30/2016)</h1>
 
<div class="entry-content">
 
<div class="entry-content">
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<b>Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/>
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The mutations of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately.
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 +
    <b>Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/>
 +
The mutants of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately.
  
 
 
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<!------------------------------------week1 end------------------------------------------------>
  
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<p> <a href="https://www.camarts.cn/archives/4252.html" class="more-link">查看Team Tianjin全部实验 <span class="meta-nav">&rsaquo;</span></a></p>
 
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<span class="sep">发表于 </span>2016 年 1 月 28 日<span class="sep"> | </span>
 
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<span id="March" class="entry-utility-prep entry-utility-prep-cat-links">发表在</span> <a href="https://www.camarts.cn/archives/category/%e8%a5%bf%e5%8c%97%e8%a1%8c" rel="category tag">春野樱</a> </span>
 
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<span class="entry-utility-prep entry-utility-prep-tag-links">标签有</span> <a href="https://www.camarts.cn/archives/tag/%e5%b8%83%e5%b0%94%e6%b4%a5" rel="tag">春野樱</a>、<a href="https://www.camarts.cn/archives/tag/xinjiang" rel="tag">新疆</a>、<a href="https://www.camarts.cn/archives/tag/%e9%98%bf%e5%8b%92%e6%b3%b0" rel="tag">漩涡鸣人</a> </span>
 
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<hr class="article">
 
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    </article><!-- #post-4252 -->
 
  
  
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<!------------------------------------week2 start------------------------------------------------>
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<div id="Week2"></div>
 
<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
<header class="entry-header">
 
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<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week2(8/15/2016-8/21/2016)</a></h1>
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<h1 class="entry-title">Week2(8/31/2016-9/6/2016)</h1>
 
</header><!-- .entry-header -->
 
</header><!-- .entry-header -->
  
 
<div class="entry-content">
 
<div class="entry-content">
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<p><li> XbaⅠ&SacⅠdouble restriction endonuclease digestion in <i>CFP</i> gene.</li><br />
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<div class="note-content2">
 +
<p><li> XbaⅠ&SacⅠdouble restriction endonuclease digestion in <i>CFP</i> gene.</li><br />
 
<li>XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1<br/>
 
<li>XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1<br/>
 
Bands as expected(760bp&3000bp) in agarose gel , gel purification of  the digested products(3000bp).
 
Bands as expected(760bp&3000bp) in agarose gel , gel purification of  the digested products(3000bp).
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Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of  E.coli  with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.
 
Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of  E.coli  with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.
 
</li><br />
 
</li><br />
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<li>Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.<br/>
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−
<li>Plasmid isolation of pRset_CFP-1-M154L.<br/>
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<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/5/55/T--Tianjin--cell-free_note-1.png"  alt="T--Tianjin--cell-free_note-1" width="800" height="533">
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/5/55/T--Tianjin--cell-free_note-1.png"  alt="T--Tianjin--cell-free_note-1" width="800" height="533">
 +
 +
<li>Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.</li><br/>
 +
<li>Plasmid isolation of pRset_CFP-1-M154L.</li><br/>
  
 
</p>
 
</p>
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<p> <a href="https://www.camarts.cn/archives/4252.html" class="more-link">查看Team Tianjin全部实验 <span class="meta-nav">&rsaquo;</span></a></p>
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<span class="sep">发表于 </span>2016 年 1 月 28 日<span class="sep"> | </span>
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<span class="entry-utility-prep entry-utility-prep-cat-links">发表在</span> <a href="https://www.camarts.cn/archives/category/%e8%a5%bf%e5%8c%97%e8%a1%8c" rel="category tag">春野樱</a> </span>
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<span class="entry-utility-prep entry-utility-prep-tag-links">标签有</span> <a href="https://www.camarts.cn/archives/tag/%e5%b8%83%e5%b0%94%e6%b4%a5" rel="tag">春野樱</a>、<a href="https://www.camarts.cn/archives/tag/xinjiang" rel="tag">新疆</a>、<a href="https://www.camarts.cn/archives/tag/%e9%98%bf%e5%8b%92%e6%b3%b0" rel="tag">漩涡鸣人</a> </span>
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<span class="total-pictures"><a href="https://www.camarts.cn/archives/4252.html">5 张图片</a></span>
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        </footer><!-- #entry-meta -->
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<!------------------------------------week2 end------------------------------------------------>
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<!------------------------------------week3 start------------------------------------------------>
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<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week3(8/29/2016-9/4/2016)</a></h1>
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<div id="Week3"></div>
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<h1 class="entry-title">Week3(9/7/2016-9/13/2016)</h1>
 
<div class="entry-content">
 
<div class="entry-content">
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<p><li>Our strategy is to exchange the site-directed mutation PETase-M154L for the other 21 mutations and 1 wild type, separately.<br/>
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 +
<div class="note-content3">
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 +
<p><li>Our strategy is to exchange the site-directed mutant PETase-M154L for the other 21 mutants and 1 wild type, separately.<br/>
 
XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1-M154L<br/>
 
XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1-M154L<br/>
 
No expected bands(3600bp&750bp) in agarose gel. We don’t find the reason why the plasmid constuced can’t be cut into the two expected bands, so we decided to change the initial strategy.  
 
No expected bands(3600bp&750bp) in agarose gel. We don’t find the reason why the plasmid constuced can’t be cut into the two expected bands, so we decided to change the initial strategy.  
 
</li><br/>
 
</li><br/>
−
<li>Ligaion of digested pRset_CFP-1, digested <i>CFP<i/> gene and the rest 22 digested gene interested (including  21 PETase site-directed mutations geneand 1 wild type ) separately in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-R90I (R90T, R90A, S92A, Q119A, M154A, W159H, W159M, N205L, N205V, S207T, S207A, I208V, S238F, S238V,A240P, S242I.)</li><br/>
+
<li>Ligaion of digested pRset_CFP-1, digested <i>CFP</i> gene and the rest 22 digested gene interested (including  21 PETase site-directed mutants geneand 1 wild type ) separately in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-R90I (R90T, R90A, S92A, Q119A, M154A, W159H, W159M, N205L, N205V, S207T, S207A, I208V, S238F, S238V,A240P, S242I.)</li><br/>
 
<li>Transformation</li>
 
<li>Transformation</li>
 
<li> Plasmid isolation</li>
 
<li> Plasmid isolation</li>
 
<li> Verification</li>
 
<li> Verification</li>
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</p> 
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<span class="sep">发表于 </span>2016 年 1 月 28 日<span class="sep"> | </span>
 
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<span class="entry-utility-prep entry-utility-prep-cat-links">发表在</span> <a href="https://www.camarts.cn/archives/category/%e8%a5%bf%e5%8c%97%e8%a1%8c" rel="category tag">春野樱</a> </span>
 
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<span class="entry-utility-prep entry-utility-prep-tag-links">标签有</span> <a href="https://www.camarts.cn/archives/tag/%e5%b8%83%e5%b0%94%e6%b4%a5" rel="tag">春野樱</a>、<a href="https://www.camarts.cn/archives/tag/xinjiang" rel="tag">新疆</a>、<a href="https://www.camarts.cn/archives/tag/%e9%98%bf%e5%8b%92%e6%b3%b0" rel="tag">漩涡鸣人</a> </span>
 
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week4(9/5/2016-9/11/2016)</a></h1>
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<div class="entry-content">
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<p><li>Colonies containing gene <i>13</i> were used to inoculate overnight cultures.</li>
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<li>Plasmids were isolated using a miniprep kit.</li>
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<b>Amplification of <i>13</i> with Q5 High-Fidelity DNA Polymerase out of E.coli </b><br/>
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<div class="note-content3">
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<li><i>13</i> amplification at 65.0°C with 13.rev/fwd primes</li>
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<!------------------------------------week3 end------------------------------------------------>      
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PCR worked, positive control worked, no amplification of <i>13</i></li>
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<li>The fragments of <i>13</i> were purified with PCR Purification Kit.</li>
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<li><i>13</i> gene fragment was phosphorylated.</li>
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<h1 class="entry-title">Week4(9/14/2016-9/20/2016)</h1>
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<div class="entry-content">
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<b>Insertion of Ni promoter and ligation of <i>13-19-15</i></b><br/>
 
  
 
<div class="note-content4">
 
<div class="note-content4">
  
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<b>Ⅱ. Expression of the plasmids constracted before</b>
  
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<li>Ni inducible promoter was ligated into pCPC-3301 vector.</li>
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<li>We choosed 4 tubes of plasmid to express in the cell-free system firstly.<br/>
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<li>Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
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The protocol of the cell-free protein synthesis system(50 µL) we used :<br/>
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<li>Single colonies were obtained by plating.</li>
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MQ H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;7.9µL<br/>
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<li>A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.</li>
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Feeding buffer&nbsp;&nbsp;&nbsp;&nbsp;25µL<br/>
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Two of the successful ones were used to inoculate overnight cultures.</li>
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Mg2+ solution&nbsp;&nbsp;&nbsp;&nbsp;1.1µL<br/>
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<li>A colony PCR of pT-13-19-15 was performed with 7 colonies.</li>
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Gene( plasmid as template)&nbsp;&nbsp;1µL<br/>
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  Two of the successful ones were used to inoculate overnight cultures.</li>
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Lysate&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;15µL<br/>
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<li>Two kinds of plasmids were isolated using a miniprep kit.</li>
+
(PS: The details of the system are not available.)<br/>
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</li>
  
 +
<b>Systems were expressed in 96-well plates and put the 96-well plates into the ELIASA (microplate reader) at 30℃ for 12 hours.</b><br/>
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<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cell-free_note-2.png"  alt="T--Tianjin--cell-free_note-2" width="800" height="533">
  
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<li><b>Parallel experiments for expression in CFPS system</b></li><br/>
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<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png"  alt="T--Tianjin--cf-m2" height="250px">
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<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/36/T--Tianjin--cf-m12.png"  alt="T--Tianjin--cf-m12"height="250px">
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<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d2/T--Tianjin--cf-m13.png"  alt="T--Tianjin--cf-m13" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/35/T--Tianjin--cf-m14.png"  alt="T--Tianjin--cf-m14" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b3/T--Tianjin--cf-m15.png"  alt="T--Tianjin--cf-m15" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/2/2c/T--Tianjin--cf-m16.png"  alt="T--Tianjin--cf-m16" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/91/T--Tianjin--cf-m17.png"  alt="T--Tianjin--cf-m17" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3f/T--Tianjin--cf-m18.png"  alt="T--Tianjin--cf-m18" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/a/ad/T--Tianjin--cf-m19.png"  alt="T--Tianjin--cf-m19" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/0/03/T--Tianjin--cf-m20.png"  alt="T--Tianjin--cf-m20" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b9/T--Tianjin--cf-m21.png"  alt="T--Tianjin--cf-m21" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cf-m22.png"  alt="T--Tianjin--cf-m22" height="250px">
 +
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3e/T--Tianjin--cf-m-no.png"  alt="T--Tianjin--cf-m-no"height="250px">
  
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<p>  
 +
<b>The figures above show that the plasmids can express in the CFPS system, although the results of the parallel experimennts indicate that the CFPS system is unstable.</b>
 +
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<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week5(9/12/2016-9/18/2016)</a></h1>
 
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<div class="entry-content">
 
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<p>
 
  
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<li>Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.</li>
 
−
<b>The sequencing results for both of them were error.</b>
 
−
<li>Colonies containing Ni inducible promoter were used to inoculate overnight cultures.</li>
 
−
<li>PCR was performed to check if the gene fragments were ligated correctly.</li>
 
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    13_ fwd and 15_rev on pT-13-19-15<br/>
 
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    Gel electrophoresis showed that it failed.<br/>
 
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<li>Plasmids pCPC-3031-Ni were isolated using a miniprep kit.</li>
 
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<li>Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
 
−
1.13_fwd and 13_rev on pT-13-19-15<br/>
 
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2.19_fwd and 19_rev on pT-13-19-15<br/>
 
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3.15_fwd and 15_rev on pT-13-19-15<br/>
 
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4.13_fwd and 19_rev on pT-13-19-15<br/>
 
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5.19_fwd and 15_rev on pT-13-19-15<br/>
 
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6.13_ wd and 15_rev on pT-13-19-15<br/>
 
−
    <b>The fourth and sixth ones were not successful.</b><br/>
 
−
<li><b>Repetition:</b>  Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
 
−
<li><b>Repetition:</b> Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
 
−
1.13_fwd and 13_rev on pT-13-19-15<br/>
 
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2.13_fwd and 19_rev on pT-13-19-15<br/>
 
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<b>The second one was failed.</b>
 
  
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<a href="https://www.camarts.cn/archives/4252.html"><img class="aligncenter size-full wp-image-4256" src="http://static.camarts.cn/images/spinner-1600.gif"  alt="IMG_2956" width="800" height="533" srcset="images/img_1.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /><noscript><img class="aligncenter size-full wp-image-4256" src="http://img.camarts.cn/2016/01/IMG_2956.jpg?imageView/2/w/800/h/800/q/90" alt="IMG_2956" width="800" height="533" srcset="http://img.camarts.cn/2016/01/IMG_2956.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /></noscript></a></p>
 
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<!------------------------------------week4 end------------------------------------------------>
  
  
  
  
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<!------------------------------------week5 start------------------------------------------------>
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<div id="Week5"></div>
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<h1 class="entry-title">Week5(9/21/2016-9/27/2016)</h1>
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<div class="entry-content">
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<div class="note-content5">
  
 +
<b>Ⅲ.Detection of Enzyme Activity</b>
 +
<p>
  
 +
<li>Substrate: 1mM pNPA  Acetonitrile solution.<br/>
 +
  10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/>
 +
  This attempt of detection failed because our enzyme precipitated in acetonitrile.
 +
</li>
  
 +
<li>Subsrtrate: 1mM pNPA Tris-HCl buffer.<br/>
 +
10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/>
 +
This attempt of detection failed, too. Because we found that Tris can react with pNPA , and pNPA would be transfered to pNP, too.
 +
</li></p>
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        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<header class="entry-header">
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<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week6(9/19/2016-9/25/2016)</a></h1>
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</header><!-- .entry-header -->
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<div class="entry-content">
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<p>
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<li>Medium preparation :BG-11.</li>
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<li>19_ fwd and 15_rev were used to amplify <i>19-15</i>.</li>
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Gel electrophoresis showed that amplification of fragments was successfull.</li>
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<li>Ligated <i>13</i> and <i>19-15</i> via overlap PCR.</li>
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−
This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.</li>
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<li>Restriction digest on pCPC-3031-Ni with Sac I.</li>
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<li>The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.</li>
+
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<li>A colony PCR of pT-13-19-15 was performed with 12 colonies.</li>
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Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.</li>
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−
<li><i>13-19-15</i> gene fragment was phosphorylated.</li>
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<li>Phosphorylated <i>13-19-15</i> was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.</li>
+
−
 
+
−
<li>Plasmids pT-13-19-15 were isolated using a miniprep kit.</li>
+
−
<li>A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.</li>
+
−
Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.</li>
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<!------------------------------------week6 start------------------------------------------------>       
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<div id="Week6"></div>
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        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
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<h1 class="entry-title">Week6(9/28/2016-10/2/2016)</h1>
 +
</header><!-- .entry-header -->
  
 +
<div class="entry-content">
 +
  
 +
<div class="note-content6">
  
 +
<p>
 +
<li>Substrate:0.2mM pNPA water solution.<br/>
 +
10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/>
 +
After static reaction at 39℃ for 8 hours, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in 400nm.
 +
</li>
  
 +
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/f/f4/T--Tianjin--cell-free_note-3.png"  alt="T--Tianjin--cf-m-no" width="800" height="533">
  
 +
<b>The control group in the image above indicate that something else in the CFPS system can cause characteristic adsorption peak except in 400nm with the sunstrate.</b><br/><br/><br/>
  
 +
<li>Substrate:PET.<br/>
 +
  PET was been put into 20 times diluted Enzyme solution(unpurified).<br/>
 +
After static reaction at 39℃ for 5 days, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm. Besides, we also deteected the product by Multispectral Scanning.
 +
</li></p>
  
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Please visit our Result part for more detailed result.
  
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<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week7(9/26/2016-10/2/2016)</a></h1>
 
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−
<div class="entry-content">
 
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<p>
 
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<li>Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.</li>
 
−
<b>The sequencing results for them were correct.</b>
 
−
 
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<br />
 
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<a href="https://www.camarts.cn/archives/4252.html"><img class="aligncenter size-full wp-image-4256" src="http://static.camarts.cn/images/spinner-1600.gif"  alt="IMG_2956" width="800" height="533" srcset="images/img_1.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /><noscript><img class="aligncenter size-full wp-image-4256" src="http://img.camarts.cn/2016/01/IMG_2956.jpg?imageView/2/w/800/h/800/q/90" alt="IMG_2956" width="800" height="533" srcset="http://img.camarts.cn/2016/01/IMG_2956.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /></noscript></a></p>
 
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Latest revision as of 14:07, 19 October 2016

TEAM TIANJIN

LOGO


Team Tianjin-Attribution

Notes For CFPS system

Week1(8/24/2016-8/30/2016)

Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)
The mutants of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately.

  • Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1(with AmpR) and plating on solid LB culture medium with Amp.
  • Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.
  • Plasmid isolation of pRset_CFP-1.
  • Primers were designed for the target (CFP gene) within the plasmid pRset_CFP-1.
  • PCR with Q5-Polymerase to amplify CFP gene out of plasmid pRset_CFP-1 and add Restriction Enzyme cutting sites(XbaⅠ&SacⅠ) to it’s ends(5’&3’) separately.
    Bands as expected(about 760bp) in agarose gel , gel purification of the PCR products.

    • Show More

    Week2(8/31/2016-9/6/2016)

  • XbaⅠ&SacⅠdouble restriction endonuclease digestion in CFP gene.

  • XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1
    Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp).

  • Ligaion of digested pRset_CFP-1, digested CFP gene and digested PETase(M154L) gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.
    Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.
    Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.

    • T--Tianjin--cell-free_note-1
  • Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

  • Plasmid isolation of pRset_CFP-1-M154L.

    • Show More

    Week3(9/7/2016-9/13/2016)

  • Our strategy is to exchange the site-directed mutant PETase-M154L for the other 21 mutants and 1 wild type, separately.
    XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1-M154L
    No expected bands(3600bp&750bp) in agarose gel. We don’t find the reason why the plasmid constuced can’t be cut into the two expected bands, so we decided to change the initial strategy.

  • Ligaion of digested pRset_CFP-1, digested CFP gene and the rest 22 digested gene interested (including 21 PETase site-directed mutants geneand 1 wild type ) separately in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-R90I (R90T, R90A, S92A, Q119A, M154A, W159H, W159M, N205L, N205V, S207T, S207A, I208V, S238F, S238V,A240P, S242I.)

  • Transformation
  • Plasmid isolation
  • Verification

    • Show More

    Week4(9/14/2016-9/20/2016)

    Ⅱ. Expression of the plasmids constracted before
  • We choosed 4 tubes of plasmid to express in the cell-free system firstly.
    The protocol of the cell-free protein synthesis system(50 µL) we used :
    MQ H2O     7.9µL
    Feeding buffer    25µL
    Mg2+ solution    1.1µL
    Gene( plasmid as template)  1µL
    Lysate     15µL
    (PS: The details of the system are not available.)
    • Systems were expressed in 96-well plates and put the 96-well plates into the ELIASA (microplate reader) at 30℃ for 12 hours.
    T--Tianjin--cell-free_note-2
  • Parallel experiments for expression in CFPS system

    • T--Tianjin--cf-blank T--Tianjin--cf-m1 T--Tianjin--cf-m2 T--Tianjin--cf-m3 T--Tianjin--cf-m4 T--Tianjin--cf-m5 T--Tianjin--cf-m6 T--Tianjin--cf-m7 T--Tianjin--cf-m8 T--Tianjin--cf-m9 T--Tianjin--cf-m10 T--Tianjin--cf-m11 T--Tianjin--cf-m12 T--Tianjin--cf-m13 T--Tianjin--cf-m14 T--Tianjin--cf-m15 T--Tianjin--cf-m16 T--Tianjin--cf-m17 T--Tianjin--cf-m18 T--Tianjin--cf-m19 T--Tianjin--cf-m20 T--Tianjin--cf-m21 T--Tianjin--cf-m22 T--Tianjin--cf-m-no

    The figures above show that the plasmids can express in the CFPS system, although the results of the parallel experimennts indicate that the CFPS system is unstable.

    Show More

    Week5(9/21/2016-9/27/2016)

    Ⅲ.Detection of Enzyme Activity

  • Substrate: 1mM pNPA Acetonitrile solution.
    10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
    This attempt of detection failed because our enzyme precipitated in acetonitrile.
  • Subsrtrate: 1mM pNPA Tris-HCl buffer.
    10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
    This attempt of detection failed, too. Because we found that Tris can react with pNPA , and pNPA would be transfered to pNP, too.

    • Show More

    Week6(9/28/2016-10/2/2016)

  • Substrate:0.2mM pNPA water solution.
    10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
    After static reaction at 39℃ for 8 hours, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in 400nm.
    • T--Tianjin--cf-m-no The control group in the image above indicate that something else in the CFPS system can cause characteristic adsorption peak except in 400nm with the sunstrate.


  • Substrate:PET.
    PET was been put into 20 times diluted Enzyme solution(unpurified).
    After static reaction at 39℃ for 5 days, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm. Besides, we also deteected the product by Multispectral Scanning.

    • Please visit our Result part for more detailed result.
    Show More
    info_outline
    Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release.     — 2016 iGEM Team Tianjin

    Team Tianjin Sponsor Alltech
    Team Tianjin Sponsor GenScript
    Team Tianjin Sponsor SynbioTech