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

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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>
 
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         <h1 class="entry-title">Week1(8/1/2016-8/7/2016)</h1>
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         <h1 class="entry-title">Week1(8/24/2016-8/30/2016)</h1>
 
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<b>Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/>
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    <b>Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/>
 
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.
 
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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<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>
 
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<p><li> XbaⅠ&SacⅠdouble restriction endonuclease digestion in <i>CFP</i> gene.</li><br />
 
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<li>XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1<br/>
 
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Bands as expected(760bp&3000bp) in agarose gel , gel purification of  the digested products(3000bp).
 
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</li><br />
 
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<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/>
 
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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/>
 
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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.
 
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</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">
 
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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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<li>We linked the remained cut <b><i>CpxR-RFP</i></b> fragment into the skeleton and then transformed the recombinant <b><i>pUC57</i></b> and the <b><i>pET21a</i></b> into<b><i> E.coli</i></b> at the same time.</li>
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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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<li>The transformation last night turned to be a failure. We tried it again.</li>
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<li>The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.</li>
 +
<li>We finally gave up the former design and decided to link the<b><i> PETase</i></b> gene into the plasmid<b><i> pUC19</i></b>. However, we did not have the key enzyme <b><i>Sal1</i></b> so we started to construct the TPA positive feedback system.</li>
 +
<li>We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the <b><i>TPA-sensing leader sequence</i></b>,<b><i> PGK1 promoter</i></b>, <b><i>CYC1 terminator</i></b>, <b><i>RFP gene</i></b>, TPA regulation protein gene (<b><i>tpaR</i></b>), TPA transporting protein gene (<b><i>tpaK</i></b>). Then we cut the fragments above and plasmid <b><i>pRS413</i></b>, <b><i>pRS415</i></b>, and <b><i>pYES2</i></b> with corresponding enzymes and recycled the fragments from agarose gel.</li>
 +
<li>We linked the fragments together by this way:<br/>
 +
<b><i>1. pYES2-leader-PGK1-RFP.<br/>
 +
2. pRS413-PGK1-tpaK-CYC1.<br/>
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3. pRS415-PGK1-tpaR-CYC1</i></b>
 +
</li> 
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<li>Then we used PCR to verify the success and all of the plasmids were correctly constructed. Then we transformed the there plasmids into <b><i>Saccharomyces cerevisiae</i></b>.
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</li>
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<li>The key enzyme <b><i>Sal1</i></b> arrived and we isolate the plasmid <b><i>pET21a</i></b>. Then we use <b><i>BamH1</i></b> and <b><i>Sal1</i></b> to cut both plasmid and <b><i>PETase</i></b> gene, then linked them together and transformed the recombinant plasmid into <b><i>E.coli</i></b>.</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"><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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<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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XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1-M154L<br/>
 
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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.
 
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</li><br/>
 
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<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/>
 
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<li>Transformation</li>
 
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<li> Plasmid isolation</li>
 
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<li> Verification</li>
 
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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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<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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<h1 class="entry-title">Week3(9/7/2016-9/13/2016)</h1>
 
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<b>Ⅱ. Expression of the plasmids constracted before<b>
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<p>
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<li>We choosed 4 tubes of plasmid to express in the cell-free system firstly.<br/>
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The protocol of the cell-free protein synthesis system(50 µL) we used :<br/>
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MQ H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;7.9µL<br/>
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Feeding buffer&nbsp;&nbsp;&nbsp;&nbsp;25µL<br/>
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Mg2+ solution&nbsp;&nbsp;&nbsp;&nbsp;1.1µL<br/>
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Gene( plasmid as template)&nbsp;&nbsp;1µL<br/>
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Lysate&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;15µL<br/>
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(PS: the details of the system are not available)<br/>
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<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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<li><b>Parallel experiments for expression in CFPS system<b></li><br/>
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<li>We cultured the transformed<b><i> E.coli</i></b> and isolated the plasmid. Then we use PCR to amplify the whole fragment in <b><i>pET21a</i></b> from <b><i>T7 promoter</i></b> to <b><i>T7 terminator</i></b>. Then we recycled this fragment from agarose gel.</li>
 +
<li>The transformed <b><i>Saccharomyces cerevisiae</i></b> had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.</li>
 +
<li>We cut the <b><i>T7 promoter-PETase gene-T7 terminator</i></b> fragment with enzymes <b><i>EcoR1</i></b> and <b><i>Sac1</i></b>. Then we linked it to the already cut plasmid <b><i>pUC19</i></b> (cut in August 28th). Then we transformed the recombinant plasmid into<b><i> E.coli</i></b>.</li>
 +
<li>We cultured the transformed <b><i>Saccharomyces cerevisiae</i></b> into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:<br/>
 +
1. Group 1: did not add TPA.<br/>
 +
2. Group 2: added 1000μL TPA standard solution.<br/>
 +
3. Group 3: added 100μL TPA standard solution.<br/>
 +
4. Group 4: added 10μL TPA standard solution.<br/>
 +
5.Group 5: added 1μL TPA standard solution.
 +
</li>
 +
<li>We cultured the transformed <b><i>E.coli</i></b> into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of <b><i>PETase</i></b> gene.</li>
 +
<li>We first detected the red fluorescence of <b><i>E.coli</i></b>, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.</li>
 +
<li>The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.</li>
 +
<li>We doubted that it might be the<b><i> RFP</i></b> in the kit was useless. We isolated the <b><i>pET21a</i></b> and used PCR to amplify the <b><i>RFP </i></b>gene.</li>         
 +
<li>We cut the <b><i>RFP</i></b> gene and <b><i>pET21a</i></b> with enzymes <b><i>Xba1</i></b> and <b><i>Sac1</i></b>, then we linked them and transformed it into <b><i>E.coli</i></b>.</li>       
 +
<li>We cultured the transformed <b><i>E.coli</i></b> and added IPTG to induce the expression of<b><i> RFP</i></b>, and this time the red fluorescence was clear enough that could be seen directly.</li> 
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<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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<h1 class="entry-title">Week4(9/14/2016-9/20/2016)</h1>
 
<div class="entry-content">
 
<div class="entry-content">
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<b>Ⅲ.Detection of Enzyme Activity<b>
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<p>
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<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.
 
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</li>
 
  
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<li>Subsrtste: 1mM pNPA Tris-HCl buffer.<br/>
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<div class="note-content4">
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10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/>
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This attempt of detection failed, too. Because we found that Tris can react with pNPA , and pNPA would be transfered to pNP, too.
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 +
<li>We started to construct another regulation way, the <b><i>E.coli</i></b> lysis regulation pathway. We first used colony PCR to obtain the <b><i>ddpX</i></b> gene from the <b><i>E.coli</i></b> genome and recycled the <b><i>ddpX</i></b> from the agarose gel.</li>
  
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<li>We found that there was no enzyme cleavage site between the<b><i> CpxR</i></b> promoter and <b><i>RFP</i></b> gene in the part we use. We had to design the primers and amplified the <b><i>CpxR</i></b> promoter by PCR.</li>
 +
<li>We used PCR to amplify the<b><i> CpxR</i></b> promoter. Then we recycled it from agarose gel.</li>
 +
<li>We cut the<b><i> CpxR</i></b> promoter with enzymes <b><i>Xba1</i></b> and<b><i> BamH1</i></b>, <b><i>ddpX</i></b> gene with enzymes <b><i>BamH1</i></b> and<b><i> EcoR1</i></b>, first batch of <b><i>pET21a</i></b> with<b><i> Xba1</i></b> and<b><i> EcoR1</i></b>, second batch of <b><i>pET21a</i></b> with<b><i> BamH1</i></b> and <b><i>EcoR1</i></b>.</li>
 +
<li>Then we linked these fragment in the following two ways:<br/>
 +
<b><i>1. pET21a-CpxR-ddpX.<br/>
 +
2. pET21a-ddpX.</i></b>
 +
</li>          
 
          
 
          
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<h1 class="entry-title">Week5(9/21/2016-9/27/2016)</h1>
 
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<li>Substrate:0.2mM pNPA water solution.<br/>
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−
10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/>
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−
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.
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</li>
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<li>Substrate:PET.<br/>
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  PET was been put into 20 times diluted Enzyme solution(unpurified).<br/>
+
−
After static reaction at 39℃ for 48 hours, 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 system by Multispectral Scanning.
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<div class="note-content5">
  
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<li>We used PCR to amplify the whole fragments in<b><i> pET21a</i></b> (from <b><i>CpxR </i></b>to <b><i>T7 terminator</i></b>). However, the band in the agarose gel was disperse so that we were unable to recycle it.</li>
  
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+
<li>We used colony PCR to verify if the <b><i>pET21a</i></b> had been correctly constructed, the result is yes.</li>
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<li>We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.</li>
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<li>We cultured the <b><i>E.coli</i></b> transformed into the <b><i>pET21a-ddpX</i></b> fragment and detect the OD600 in order to verify the lysis effection of <b><i>ddpX</i></b>. </li>
−
 
+
<li>Considering the <b><i>pYES2</i></b> is multicopy plasmid so that the copy number would affect the <b><i>RFP</i></b> expression level, we decided to change the <b><i>pYES2</i></b> to single-copy plasmid <b><i>pRS416</i></b>. Since the <b><i>pRS416</i></b> does not have terminator in its backbone, we used PCR to amplify the <b><i>CYC1</i></b> terminator from plasmid <b><i>pYES2</i></b>.</li>           
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<li>We cut the <b><i>pYES2</i></b> with enzyme <b><i>Hind3</i></b> and <b><i>EcoR1</i></b>, <b><i>CYC1</i></b> with<b><i> EcoR1</i></b> and <b><i>Sal1</i></b>,<b><i> pRS416</i></b> with <b><i>Hind3</i></b> and<b><i> Sal1</i></b>. Then we linked the three part together.</li>       
 +
<li>We transformed the three plasmids into <b><i>Saccharomyces cerevisiae</i></b> together. </li>
 +
<li>The new primers using to amplify the<b><i> CpxR-ddpX-T7</i></b> terminator fragment arrived and we redid the PCR. However, the disperse band was still existed. </li>
 +
<li>The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate. </li>
 +
     
 
          
 
          
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<h1 class="entry-title">Week6(9/28/2016-10/2/2016)</h1>
 
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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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<li>We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with <b><i>PETase</i></b> gene and <b><i>CpxR-RFP</i></b> fragment showed the deepest red.</li>
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Revision as of 02:36, 4 October 2016

TEAM TIANJIN

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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 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.

  • 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.

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

  • We linked the remained cut CpxR-RFP fragment into the skeleton and then transformed the recombinant pUC57 and the pET21a into E.coli at the same time.
  • The transformation last night turned to be a failure. We tried it again.
  • The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.
  • We finally gave up the former design and decided to link the PETase gene into the plasmid pUC19. However, we did not have the key enzyme Sal1 so we started to construct the TPA positive feedback system.
  • We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the TPA-sensing leader sequence, PGK1 promoter, CYC1 terminator, RFP gene, TPA regulation protein gene (tpaR), TPA transporting protein gene (tpaK). Then we cut the fragments above and plasmid pRS413, pRS415, and pYES2 with corresponding enzymes and recycled the fragments from agarose gel.
  • We linked the fragments together by this way:
    1. pYES2-leader-PGK1-RFP.
    2. pRS413-PGK1-tpaK-CYC1.
    3. pRS415-PGK1-tpaR-CYC1
  • Then we used PCR to verify the success and all of the plasmids were correctly constructed. Then we transformed the there plasmids into Saccharomyces cerevisiae.
  • The key enzyme Sal1 arrived and we isolate the plasmid pET21a. Then we use BamH1 and Sal1 to cut both plasmid and PETase gene, then linked them together and transformed the recombinant plasmid into E.coli.
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    Week3(9/7/2016-9/13/2016)

  • We cultured the transformed E.coli and isolated the plasmid. Then we use PCR to amplify the whole fragment in pET21a from T7 promoter to T7 terminator. Then we recycled this fragment from agarose gel.
  • The transformed Saccharomyces cerevisiae had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.
  • We cut the T7 promoter-PETase gene-T7 terminator fragment with enzymes EcoR1 and Sac1. Then we linked it to the already cut plasmid pUC19 (cut in August 28th). Then we transformed the recombinant plasmid into E.coli.
  • We cultured the transformed Saccharomyces cerevisiae into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:
    1. Group 1: did not add TPA.
    2. Group 2: added 1000μL TPA standard solution.
    3. Group 3: added 100μL TPA standard solution.
    4. Group 4: added 10μL TPA standard solution.
    5.Group 5: added 1μL TPA standard solution.
  • We cultured the transformed E.coli into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of PETase gene.
  • We first detected the red fluorescence of E.coli, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.
  • The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.
  • We doubted that it might be the RFP in the kit was useless. We isolated the pET21a and used PCR to amplify the RFP gene.
  • We cut the RFP gene and pET21a with enzymes Xba1 and Sac1, then we linked them and transformed it into E.coli.
  • We cultured the transformed E.coli and added IPTG to induce the expression of RFP, and this time the red fluorescence was clear enough that could be seen directly.
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    Week4(9/14/2016-9/20/2016)

  • We started to construct another regulation way, the E.coli lysis regulation pathway. We first used colony PCR to obtain the ddpX gene from the E.coli genome and recycled the ddpX from the agarose gel.
  • We found that there was no enzyme cleavage site between the CpxR promoter and RFP gene in the part we use. We had to design the primers and amplified the CpxR promoter by PCR.
  • We used PCR to amplify the CpxR promoter. Then we recycled it from agarose gel.
  • We cut the CpxR promoter with enzymes Xba1 and BamH1, ddpX gene with enzymes BamH1 and EcoR1, first batch of pET21a with Xba1 and EcoR1, second batch of pET21a with BamH1 and EcoR1.
  • Then we linked these fragment in the following two ways:
    1. pET21a-CpxR-ddpX.
    2. pET21a-ddpX.
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    Week5(9/21/2016-9/27/2016)

  • We used PCR to amplify the whole fragments in pET21a (from CpxR to T7 terminator). However, the band in the agarose gel was disperse so that we were unable to recycle it.
  • We used colony PCR to verify if the pET21a had been correctly constructed, the result is yes.
  • We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.
  • We cultured the E.coli transformed into the pET21a-ddpX fragment and detect the OD600 in order to verify the lysis effection of ddpX.
  • Considering the pYES2 is multicopy plasmid so that the copy number would affect the RFP expression level, we decided to change the pYES2 to single-copy plasmid pRS416. Since the pRS416 does not have terminator in its backbone, we used PCR to amplify the CYC1 terminator from plasmid pYES2.
  • We cut the pYES2 with enzyme Hind3 and EcoR1, CYC1 with EcoR1 and Sal1, pRS416 with Hind3 and Sal1. Then we linked the three part together.
  • We transformed the three plasmids into Saccharomyces cerevisiae together.
  • The new primers using to amplify the CpxR-ddpX-T7 terminator fragment arrived and we redid the PCR. However, the disperse band was still existed.
  • The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate.
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    Week6(9/28/2016-10/2/2016)

  • We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with PETase gene and CpxR-RFP fragment showed the deepest red.
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    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

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