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| | {{:Team:Tianjin/Templates/MaterialTheme|}} | | {{:Team:Tianjin/Templates/MaterialTheme|}} |
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| − | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Note/CFPS/camart.css}} | + | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Note/R-R/camarts.css}} |
| | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Experiment/css/font.css}} | | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Experiment/css/font.css}} |
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| | <html lang="en"> | | <html lang="en"> |
| | + | <style> |
| | + | #Notes{ |
| | + | color: inherit; |
| | + | background-color: rgba(255, 255, 255, 0.1); |
| | + | } |
| | + | </style> |
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| | <head> | | <head> |
| | <meta charset="utf-8" /> | | <meta charset="utf-8" /> |
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| | <!------------------------------------------- This part should be dismissed in WIKI u | | <!------------------------------------------- This part should be dismissed in WIKI u |
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| | <div id="page-heading" class="relative"> | | <div id="page-heading" class="relative"> |
| − | <img class="ribbon-version1" src="images/ribbon-2016 - 副本.png">
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| | <header id="main-header" role="banner"> | | <header id="main-header" role="banner"> |
| − | | + | <style> |
| | + | .note-content,.note-content2,.note-content3,.note-content4,.note-content5,.note-content6,.note-content li,.note-content2 li,.note-content3 li,.note-content4 li,.note-content5 li,.note-content6 li{ |
| | + | font-family:Arial; |
| | + | font-size:18px; |
| | + | text-align:justify; |
| | + | } |
| | + | </style> |
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| − | <nav id="nav" class="nav" role="navigation">
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| − | <a href="#nav" title="显示菜单" data-ajax="false">菜单</a>
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| − | <a href="#" title="隐藏菜单" data-ajax="false">关闭</a>
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| − | <ul role="menubar">
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| − | </nav>
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| | </div> | | </div> |
| | </header> | | </header> |
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| | <div id="main"> | | <div id="main"> |
| | <div id="content" class="home blog single-author one-column content" role="main"> | | <div id="content" class="home blog single-author one-column content" role="main"> |
| − | <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"> | + | |
| | + | <!--------------------------------week1 start--------------------------------------------> |
| | + | <div id="Week1"></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"> |
| | <header class="entry-header"> | | <header class="entry-header"> |
| − | <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> | + | <div class="entry-title" align="center" >Notes For CFPS system</div> |
| | </header><!-- .entry-header --> | | </header><!-- .entry-header --> |
| − | <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> | + | <h1 class="entry-title">Week1(8/24/2016-8/30/2016)</h1> |
| | <div class="entry-content"> | | <div class="entry-content"> |
| − | <p> | + | <div class="note-content"> |
| − | <li>Cultivation: 30µL mother liquid of Synechococcus sp. PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.<br/></li>
| + | |
| − | <li>Cultivation: 30µL mother liquid of Synechococcus sp. PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.<br/></li> | + | <b>Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/> |
| − | <li>Cultivation: 30µL mother liquid of Synechococcus sp. PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.<br/></li> | + | 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. |
| − | <li>Cultivation: 30µL mother liquid of Synechococcus sp. PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.<br/></li>
| + | |
| | + | |
| | + | <p> |
| | + | <li>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.<br/></li> |
| | + | <li>Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.<br/></li> |
| | + | <li>Plasmid isolation of pRset_CFP-1.<br/></li> |
| | + | <li>Primers were designed for the target (<i>CFP</i> gene) within the plasmid pRset_CFP-1.<br/></li> |
| | + | <li>PCR with Q5-Polymerase to amplify <i>CFP</i> gene out of plasmid pRset_CFP-1 and add Restriction Enzyme cutting sites(XbaⅠ&SacⅠ) to it’s ends(5’&3’) separately.<br/> |
| | + | Bands as expected(about 760bp) in agarose gel , gel purification of the PCR products. |
| | + | <br/></li> |
| | </p> | | </p> |
| | + | |
| | + | </div> |
| | + | <a class="expand-btn">Show More</a> |
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| − | <!------------------------------------------------------外加效果-------------------------------------------->
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| | + | </div><!-- .entry-content --> |
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| − | <!--------------------------------------------------外加效果------------------------------------------------> | + | |
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| | + | <!-- <hr class="article"> |
| | + | </article> #post-4252 --> |
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| − | <a href="https://www.camarts.cn/archives/4252.html"> | + | <!------------------------------------week1 end------------------------------------------------> |
| − | <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="IMG_2956" width="800" height="533">
| + | |
| − | </a>
| + | |
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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">›</span></a></p>
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| − | </div><!-- .entry-content -->
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| − |
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| − | <footer class="entry-meta">
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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="cat-links">
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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="sep"> | </span>
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| − | <span class="tag-links">
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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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| − |
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| − | <span class="sep"> | </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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| − |
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| − | </footer><!-- #entry-meta -->
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| − |
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| − | <hr class="article">
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| − | </article><!-- #post-4252 -->
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| | | | |
| | + | <!------------------------------------week2 start------------------------------------------------> |
| | + | <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"> | | <header class="entry-header"> |
| − | <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> | + | <h1 class="entry-title">Week2(8/31/2016-9/6/2016)</h1> |
| | </header><!-- .entry-header --> | | </header><!-- .entry-header --> |
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| | <div class="entry-content"> | | <div class="entry-content"> |
| − | <p><li>Synechococcus sp PCC 7942 had no signals of life.</li><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="IMG_2956" width="800" height="533">
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| − |
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| − | </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">›</span></a></p>
| |
| − | </div><!-- .entry-content -->
| |
| − |
| |
| − | <footer class="entry-meta">
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| − |
| |
| − | <span class="sep">发表于 </span>2016 年 1 月 28 日<span class="sep"> | </span>
| |
| | | | |
| − | <span class="cat-links">
| + | <div class="note-content2"> |
| − | <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>
| + | <p><li> XbaⅠ&SacⅠdouble restriction endonuclease digestion in <i>CFP</i> gene.</li><br /> |
| − | <span class="sep"> | </span>
| + | <li>XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1<br/> |
| − | <span class="tag-links">
| + | Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp). |
| − | <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>
| + | </li><br /> |
| − |
| + | <li>Ligaion of digested pRset_CFP-1, digested <i>CFP</i> gene and digested <i>PETase(M154L)</i> gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.<br/> |
| − | <span class="sep"> | </span>
| + | 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.<br/> |
| − | <span class="total-pictures"><a href="https://www.camarts.cn/archives/4252.html">5 张图片</a></span>
| + | 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 /> |
| − | </footer><!-- #entry-meta -->
| + | |
| − |
| + | |
| − | <hr class="article">
| + | |
| − | </article>
| + | |
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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"> |
| | | | |
| | + | <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/> |
| | | | |
| − | <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">
| + | </p> |
| − | <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>
| + | </div> |
| − | <div class="entry-content">
| + | <a class="expand-btn2">Show More</a> |
| − | <p><li>pMV-G19</li>
| + | |
| − | <li>pMV-G15</li>
| + | |
| − | <li>Colonies were used to inoculate overnight cultures.</li>
| + | |
| − | <li>Plasmids were isolated using a miniprep kit.</li>
| + | |
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| − | <b>Amplification of <i>19</i> and <i>15</i> with Q5 High-Fidelity DNA Polymerase out of E.coli </b><br/>
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| − | <div class="note-content"> | + | </div><!-- .entry-content --> |
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| − | <li><i>19</i> amplification at 65.0°C with 19.rev/fwd primes.</li>
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| − | PCR worked, positive control worked, no amplification of <i>19</i>.
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| − | <li><i>15</i> amplification at 65.0°C with 15.rev/fwd primes.</li>
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| − | PCR worked, positive control worked, no amplification of <i>15</i>.
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| − | <li>A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.</li>
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| − | <li>This result was confirmed by sequencing.</li>
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| − | <li>The fragments of <i>19</i> and <i>15</i> were purified with PCR Purification Kit.</li>
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| | | | |
| − | </div>
| + | <!------------------------------------week2 end------------------------------------------------> |
| − | <a class="expand-btn">Show More</a>
| + | |
| | | | |
| | + | <!------------------------------------week3 start------------------------------------------------> |
| | + | <div id="Week3"></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"> |
| | + | <h1 class="entry-title">Week3(9/7/2016-9/13/2016)</h1> |
| | + | <div class="entry-content"> |
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| − | <b>Ligation of <i>15</i> with <i>19</i></b><br/>
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| − | <div class="note-content2"> | + | <div class="note-content3"> |
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| − | <li>We add a single 3`-adenine overhang to each end of the fragment of <i>19</i> and then purified with DNA Purification Kit.</li>
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| − | <li><i>19</i> was ligated into T vectors via TA clone and transformed into E.coli via heat shock.</li>
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| − | <li>A colony PCR was performed with five colonies.</li>
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| − | Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.</li>
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| − | Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.</li>
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| − | <li><i>15</i> gene fragment was phosphorylated.</li>
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| − | <li>Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.</li>
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| − | <li>Mono-restriction digest of pT-19 with stu I. </li>
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| − | <li>The enzyme-digested product was dephosphorylation.</li>
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| − | <li>Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.</li>
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| − | Ligation product was transformed into E.coli via heat shock.</li>
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| − | <li>A colony PCR was performed with twelve colonies.</li>
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| − | Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.</li>
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| − | These two colonies were used to inoculate overnight cultures.</li>
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| − | <li>Plasmids with correct sequence of <i>19-15</i> were isolated using a miniprep kit.</li>
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| − | <li>Mono-restriction digest of pT-19-15 with Nru I.</li>
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| − | <li>The enzyme-digested product was dephosphorylation.</li>
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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/> |
| | + | 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>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> Plasmid isolation</li> |
| | + | <li> Verification</li> |
| | + | </p> |
| | + | |
| | </div> | | </div> |
| − | <a class="expand- btn2">Show More</a> | + | <a class="expand-btn3">Show More</a> |
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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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| − | <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">›</span></a></p>
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| − | </div><!-- .entry-content -->
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| − |
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| − | <footer class="entry-meta">
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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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| − |
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| − | <span class="cat-links">
| |
| − | <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="sep"> | </span>
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| − | <span class="tag-links">
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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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| − |
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| − | <span class="sep"> | </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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| − |
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| − | </footer><!-- #entry-meta -->
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| − |
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| − | <hr class="article">
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| − | </article>
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| | + | </div><!-- .entry-content --> |
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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">
| + | |
| − | <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>
| + | |
| − | <div class="entry-content"> | + | |
| − | <p><li>Colonies containing gene <i>13</i> were used to inoculate overnight cultures.</li>
| + | |
| − | <li>Plasmids were isolated using a miniprep kit.</li>
| + | |
| − | <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> | + | <!------------------------------------week3 end------------------------------------------------> |
| − | PCR worked, positive control worked, no amplification of <i>13</i></li>
| + | |
| − | <li>The fragments of <i>13</i> were purified with PCR Purification Kit.</li>
| + | |
| − | <li><i>13</i> gene fragment was phosphorylated.</li>
| + | |
| | | | |
| − |
| + | |
| | | | |
| − | </div>
| |
| − | <a class="expand-btn3">Show More</a>
| |
| | | | |
| | + | <!------------------------------------week4 start------------------------------------------------> |
| | + | <div id="Week4"></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"> |
| | + | <h1 class="entry-title">Week4(9/14/2016-9/20/2016)</h1> |
| | + | <div class="entry-content"> |
| | + | |
| | | | |
| − | <b>Insertion of Ni promoter and ligation of <i>13-19-15</i></b><br/>
| |
| | | | |
| | <div class="note-content4"> | | <div class="note-content4"> |
| | | | |
| | + | <b>Ⅱ. Expression of the plasmids constracted before</b> |
| | | | |
| − | <li>Ni inducible promoter was ligated into pCPC-3301 vector.</li> | + | <li>We choosed 4 tubes of plasmid to express in the cell-free system firstly.<br/> |
| − | <li>Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li> | + | The protocol of the cell-free protein synthesis system(50 µL) we used :<br/> |
| − | <li>Single colonies were obtained by plating.</li> | + | MQ H2O 7.9µL<br/> |
| − | <li>A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.</li>
| + | Feeding buffer 25µL<br/> |
| − | Two of the successful ones were used to inoculate overnight cultures.</li>
| + | Mg2+ solution 1.1µL<br/> |
| − | <li>A colony PCR of pT-13-19-15 was performed with 7 colonies.</li>
| + | Gene( plasmid as template) 1µL<br/> |
| − | Two of the successful ones were used to inoculate overnight cultures.</li>
| + | Lysate 15µL<br/> |
| − | <li>Two kinds of plasmids were isolated using a miniprep kit.</li>
| + | (PS: The details of the system are not available.)<br/> |
| | + | </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/> |
| | + | <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"> |
| | | | |
| − |
| + | <li><b>Parallel experiments for expression in CFPS system</b></li><br/> |
| − |
| + | |
| − | </div> | + | |
| − | <a class="expand-btn4">Show More</a>
| + | |
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| | + | |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-blank.png" alt="T--Tianjin--cf-blank" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d0/T--Tianjin--cf-m1.png" alt="T--Tianjin--cf-m1" height="250px"> |
| | + | <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"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/34/T--Tianjin--cf-m3.png" alt="T--Tianjin--cf-m3" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/7/75/T--Tianjin--cf-m4.png" alt="T--Tianjin--cf-m4" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/be/T--Tianjin--cf-m5.png" alt="T--Tianjin--cf-m5" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m6.png" alt="T--Tianjin--cf-m6" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m7.png" alt="T--Tianjin--cf-m7" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-m8.png" alt="T--Tianjin--cf-m8" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d1/T--Tianjin--cf-m9.png" alt="T--Tianjin--cf-m9" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/6/61/T--Tianjin--cf-m10.png" alt="T--Tianjin--cf-m10" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/c/c9/T--Tianjin--cf-m11.png" alt="T--Tianjin--cf-m11" height="250px"> |
| | + | <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"> |
| | + | <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"> |
| | | | |
| − | <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> | + | <br/> |
| − | <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">›</span></a></p>
| + | |
| − | </div><!-- .entry-content -->
| + | |
| | | | |
| − | <footer class="entry-meta">
| + | <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> |
| | + | </p> |
| | + | |
| | + | |
| | | | |
| − | <span class="sep">发表于 </span>2016 年 1 月 28 日<span class="sep"> | </span>
| |
| − |
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| − | <span class="cat-links">
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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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| | + | </div> |
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| − |
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| − |
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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">
| |
| − | <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>
| |
| − | <div class="entry-content">
| |
| − | <p>
| |
| − |
| |
| − | <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>
| |
| − | 13_ fwd and 15_rev on pT-13-19-15<br/>
| |
| − | Gel electrophoresis showed that it failed.<br/>
| |
| − | <li>Plasmids pCPC-3031-Ni were isolated using a miniprep kit.</li>
| |
| − | <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/>
| |
| − | 2.19_fwd and 19_rev on pT-13-19-15<br/>
| |
| − | 3.15_fwd and 15_rev on pT-13-19-15<br/>
| |
| − | 4.13_fwd and 19_rev on pT-13-19-15<br/>
| |
| − | 5.19_fwd and 15_rev on pT-13-19-15<br/>
| |
| − | 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/>
| |
| − | 2.13_fwd and 19_rev on pT-13-19-15<br/>
| |
| − | <b>The second one was failed.</b>
| |
| − |
| |
| − |
| |
| − | <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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| − | <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">›</span></a></p>
| |
| | </div><!-- .entry-content --> | | </div><!-- .entry-content --> |
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| − | <footer class="entry-meta"> | + | |
| − |
| + | |
| − | <span class="sep">发表于 </span>2016 年 1 月 28 日<span class="sep"> | </span>
| + | |
| − |
| + | |
| − | <span class="cat-links">
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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>
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| | | | |
| | + | <!------------------------------------week4 end------------------------------------------------> |
| | | | |
| | | | |
| | | | |
| | | | |
| | + | <!------------------------------------week5 start------------------------------------------------> |
| | + | <div id="Week5"></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"> |
| | + | <h1 class="entry-title">Week5(9/21/2016-9/27/2016)</h1> |
| | + | <div class="entry-content"> |
| | + | |
| | | | |
| | + | <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> |
| | + | |
| | + | |
| | | | |
| − | <!-- #post-4252 --> | + | </div> |
| − |
| + | <a class="expand-btn5">Show More</a> |
| − | <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">
| + | |
| − | <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> | + | |
| − | </header><!-- .entry-header -->
| + | |
| − | | + | |
| − | <div class="entry-content">
| + | |
| − | <p>
| + | |
| − | <li>Medium preparation :BG-11.</li>
| + | |
| − | <li>19_ fwd and 15_rev were used to amplify <i>19-15</i>.</li>
| + | |
| − | Gel electrophoresis showed that amplification of fragments was successfull.</li>
| + | |
| − | <li>Ligated <i>13</i> and <i>19-15</i> via overlap PCR.</li>
| + | |
| − | This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.</li>
| + | |
| − | <li>Restriction digest on pCPC-3031-Ni with Sac I.</li>
| + | |
| − | <li>The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.</li>
| + | |
| − | <li>A colony PCR of pT-13-19-15 was performed with 12 colonies.</li>
| + | |
| − | Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.</li>
| + | |
| − | <li><i>13-19-15</i> gene fragment was phosphorylated.</li>
| + | |
| − | <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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| − | <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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| − | <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">›</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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| | + | <h1 class="entry-title">Week6(9/28/2016-10/2/2016)</h1> |
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| | + | <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"> |
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| | + | <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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| − | <li>Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.</li>
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| − | <b>The sequencing results for them were correct.</b>
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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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| − | <b>Notice:</b> 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 | + | <b>Notice: </b> 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 |
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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.