Difference between revisions of "Team:NEFU China/Notebook"

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    <title>Flight iGEM</title>
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<p><strong><span style="font-family:comic sans ms,cursive"><span style="font-size:28px">Monthly</span></span></strong></p>
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<span style="font-family:arial,helvetica,sans-serif"><strong><span style="font-size:18px">January</span></strong><br />
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<span style="font-size:14px">Our junior and senior students were attracted by the recruiting announcement for the iGEM 2015 in our university. After several interviews and competitive selections, our team was basically created.</span><br />
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<strong><span style="font-size:18px">February</span></strong><br />
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<span style="font-size:14px">Our team was growing. We communicated with previous iGEMers in our school to get more insights into iGEM. Meanwhile, we started to watch successful iGEM cases of previous years.</span><br />
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<strong><span style="font-size:18px">March</span></strong><br />
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<span style="font-size:14px">We wandered among wikis and gradually realized what the basic factors are for a good project. Brainstorms and weekly meet-ups promoted us to discover the quintessence of iGEM.</span><br />
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<strong><span style="font-size:18px">April</span></strong><br />
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<span style="font-size:14px">We urged ourselves to get deeper insights into those previous cases to catch their inspiration. After meet-ups with teammates and our instructors, a draft idea of a yogurt guarder came to our mind. To make sure our project was worth proceeding, we did a survey to find out how much attention people would pay to the quality of yogurt. Fortunately, we got the expected results, although we knew we still had a long way to go.</span><br />
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<span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">We primarily designed the basic structure of our project. Then we read numerous papers to make an integrated protocol of our lab work.</span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">Besides, we visited a dairy factory to learn the microbiological detection methods used in practical production of yogurt. We discovered that our method to be developed should have advantages over the traditional methods, especially in portability.&nbsp;</span></span><br />
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<span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">In addition, it was essential for us to adapt to the wet lab work. We started with laboratory safety training. With the instruction of our instructors and previous iGEMers, we learned a lot of techniques in molecular biology.</span><br />
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<strong><span style="font-size:18px">June</span></strong><br />
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<span style="font-size:14px">With efforts, our wet lab work went smoothly.</span><br />
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<span style="font-size:14px">This month, we organized public activity to popularize the knowledge of yogurt production and its quality inspection, and remind people to store yogurt properly.&nbsp;</span></span><br />
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<span style="font-family:arial,helvetica,sans-serif"><span style="font-size:14px">In addition, we did a survey to confirm the key factors which will attract customers&rsquo; attention if our yogurt guarder is developed into a real product.&nbsp;</span><br />
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<strong><span style="font-size:18px">August</span></strong><br />
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<span style="font-size:14px">On 13th-15th, we attended the summit of Central China iGEM Consortium held by Peking University. We shared our experience with other teams and really learned a lot from other teams. Here we want to express our sincere thanks to them!&nbsp;<br />
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Meanwhile, our lab works still proceeded. We encountered difficulty in the electrotransformation of bacteria, which delayed our project but was eventually resolved after multiple attempts.</span><br />
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<strong><span style="font-size:18px">September</span></strong><br />
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<span style="font-size:14px">Time flies. We seized the time to make everything done and put great efforts to the wiki building and preparation for the Giant Jamboree.</span></span></p>
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<p><strong><span style="font-family:comic sans ms,cursive"><span style="font-size:28px">Labnote</span></span></strong></p>
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          <div class="logo-container"><span class="logo">Flight iGEM</span></div>
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          <h2>Facts<em></em></h2>
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                <h2 style="text-align:center;">Attitudes towards creating a wiki</h2>
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                  <p>According to a survey conducted by us, we found that most teams agree&nbsp;the thesis that creating wiki is a difficult or tedious work to finish with high quality, especially when time is very limited. So, we&rsquo;d like to make some changes.</p>
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<p>*You can see all raw data by click <a href="http://www.wenjuan.com/r/QF3QnyK?pid=55f01be9f7405b0c34569df1&amp;vcode=d3452b2f6d3f5f4e009805e3520d4512">this</a>.</p>
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<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:20px">Isolation&nbsp;of&nbsp;genomic&nbsp;DNA&nbsp;from&nbsp;bacteria</span></span>&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<span style="font-family:arial,helvetica,sans-serif">The&nbsp;genomic&nbsp;DNA&nbsp;of&nbsp;<em>Salmonella&nbsp;enterica</em>&nbsp;subsp.&nbsp;<em>enterica&nbsp;serovar&nbsp;Typhimurium</em>&nbsp;str.&nbsp;LT2&nbsp;and<em>&nbsp;Lactobacillus&nbsp;delbrueckii</em>&nbsp;subsp.<em>&nbsp;Bulgaricus</em>&nbsp;str.&nbsp;Lb14&nbsp;were&nbsp;isolated&nbsp;using&nbsp;the&nbsp;TIANamp&nbsp;Bacteria&nbsp;DNA&nbsp;Kit&nbsp;(see&nbsp;protocol).&nbsp;The&nbsp;concentration&nbsp;of&nbsp;genomic&nbsp;DNA&nbsp;was&nbsp;measured&nbsp;with&nbsp;a&nbsp;NanoDrop&nbsp;spectrophotometer.&nbsp;</span>&nbsp;&nbsp; &nbsp; &nbsp; &nbsp;</p>
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          <div class="logo-container"><span class="logo">Flight iGEM</span></div>
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          <h2>Flight iGEM Magic<em></em></h2>
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<p><span style="font-size:20px"><span style="font-family:arial,helvetica,sans-serif">Strain&nbsp;identification</span></span></p>
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<p>About half of teams we surveyed hold the opinion that creating a wiki requires superior skills in programming (29/56), which lead them feel hard about it. We sincerely invite you to click the right arrow and discover what giant changes Flight iGEM can make.</p>
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                <div class="col-xs-6 col-md-6"><img class="himg" alt="" src="https://static.igem.org/mediawiki/2015/6/61/NEFU_China_FABE4DBA-CD83-486D-AAC2-E4B3949D76D8.png" /></div>
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<div class="col-xs-6 col-md-6">
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<p><span style="font-family:arial,helvetica,sans-serif">We&nbsp;applied&nbsp;16S&nbsp;ribosomal&nbsp;DNA&nbsp;identification&nbsp;to&nbsp;identify&nbsp;the&nbsp;strain&nbsp;of&nbsp;Salmonella&nbsp;and&nbsp;Lactobacillus&nbsp;bought&nbsp;from&nbsp;a&nbsp;biotech&nbsp;company.&nbsp;Universal&nbsp;primers&nbsp;were&nbsp;designed&nbsp;according&nbsp;to&nbsp;the&nbsp;conserved&nbsp;sequences.</span></p>
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<h1>Step 1</h1>
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<p>To create a wiki, a HTML template is necessary neither with Flight iGEM nor without Flight iGEM.</p>
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<p><span style="font-size:20px"><span style="font-family:arial,helvetica,sans-serif">A.&nbsp;Identification&nbsp;of&nbsp;Salmonella&nbsp;typhimurium</span></span></p>
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                <h1>Step 2</h1>
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<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
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<h2>Without Flight iGEM</h2>
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<img alt="" class="himg" src="https://static.igem.org/mediawiki/2015/3/30/NEFU_China_60AF68F6-696F-4A98-844E-2023C8011191.png" />
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<p>Once the template is done, other teammates can start to summarize the project, record experiments&rsquo; data by using their Word or iWork.</p>
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<p><img alt="" src="https://static.igem.org/mediawiki/2015/0/05/NEFU_China_5351A137-7EAB-4153-B27D-FDEDBACC8656.png" style="height:393px; width:700px" /></p>
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<h2>With Flight iGEM</h2>
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<img alt="" class="himg" src="https://static.igem.org/mediawiki/2015/6/6f/NEFU_China_75095857-0A36-4862-8293-8993E03284D5.png" />Flight iGEM requires you and your teammates to work online. We use CKEditor(a free, Open Source HTML text editor) to set up edit environment like Word or iWork.</div>              </div>
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                <h1>Step 3</h1>
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<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/0/08/NEFU_China_1A00253D-C888-4317-83AB-12FBD6D8F14E.png" style="height:225px; width:150px" /></p>
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<h2>Without Flight iGEM</h2>
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<img class="himg" src="https://static.igem.org/mediawiki/2015/3/3c/NEFU_China_53ABE671-6E9D-4A6A-A6CA-6C58897625F2.png" />
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<p>Without Flight iGEM, you need to upload all images and other media files which involved in your articles to the iGEM server manually.</p>
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<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;Fig 1. 1. Marker (2000bp DNA Ladder) ;&nbsp;2.<span style="font-size:16px">&nbsp;</span><span style="font-size:14px"><em>Salmonella&nbsp;typhimurium</em></span><span style="font-size:16px">&nbsp;</span>16s rRNA</span></p>
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<h2>With Flight iGEM</h2>
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<img class="himg" src="https://static.igem.org/mediawiki/2015/0/09/NEFU_China_7C064076-7096-4A36-918C-6602AC0862D3.png" />
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<p>If you use Flight iGEM, all media files(especially the images) which you insert into the article will be automatically upload to iGEM server.</p>
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                <h1>Step 4</h1>
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<p><span style="font-family:arial,helvetica,sans-serif">2.&nbsp;Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;see&nbsp;protocol)</span></p>
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<h2>Without Flight iGEM</h2>
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<img class="himg" src="https://static.igem.org/mediawiki/2015/e/e6/NEFU_China_DEE6107D-1149-460D-A6CA-F9A0FCEEBE66.png" />
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<p>You need to assign the content which converted from Word or iWork documents to the template. In this step, there will be numerous words and sentences that will be copied and pasted, which cost a lot of time and memory.</p>
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<div class="col-xs-6 col-md-6">
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<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/6c/NEFU_China_CD600935-F7B2-41DF-AE7A-1356B2D74459.png" style="height:117px; width:700px" /></p>
−
<h2>With Flight iGEM</h2>
+
−
<img class="himg" src="https://static.igem.org/mediawiki/2015/d/d5/NEFU_China_8EA8F25C-716E-480F-9DFC-B3B0B58DD2B0.png" />
+
−
<p>Flight iGEM can assign contents with template automatically, all you need to do is to select what you want to display, and then click &#39;Magic&#39;.</p>
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</div>              </div>
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            </div>
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          </div>
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        </div>
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−
        <div class="slide">
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          <div class="container">
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            <div class="raw">
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              <div class="sub-container">
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                <h1>Step 5</h1>
+
  
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<div class="col-xs-6 col-md-6">
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<p><span style="font-family:arial,helvetica,sans-serif">3.&nbsp;Transformation&nbsp;(<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a>)</span></p>
−
<h2>Without Flight iGEM</h2>
+
−
<img class="himg" src="https://static.igem.org/mediawiki/2015/0/05/NEFU_China_419F93B3-DBB5-46EF-A785-05A0D702C8F0.png" />
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−
<p>After you merged this two page, there are you should upload the &quot;code soup&quot; to the iGEM server, and this is the final step of the whole procedure.</p>
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−
</div>
+
  
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<div class="col-xs-6 col-md-6">
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<p><span style="font-family:arial,helvetica,sans-serif">4.&nbsp;Colony&nbsp;PCR</span></p>
−
<h2>With Flight iGEM</h2>
+
 
−
<img class="himg" src="https://static.igem.org/mediawiki/2015/6/6a/NEFU_China_E5C6420A-4FEF-4898-BDED-AFCAEB7E36AF.png" />
+
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/fa/NEFU_China_80678D69-C533-4A86-A51D-001697EC74D8.png" style="height:380px; width:700px" /></p>
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<p>Flight iGEM will upload your pages to the iGEM server after step 4, and the only thing you need to do is to visit your target page and check if the page is exactly what you want.</p>
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−
</div>             </div>
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<p style="text-align:center"><span style="color:rgb(0, 255, 255)"><span style="font-size:28px"><img alt="" src="https://static.igem.org/mediawiki/2015/c/ce/NEFU_China_418396AA-6D58-4C22-ADA9-5820CEC9AD9A.png" style="height:122px; width:200px" /></span></span></p>
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            </div>
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          </div>
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<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 2.&nbsp;1. Marker (DL2000) ; 2.3.4.5&nbsp;each for one single colony; 6. Negative control</span>&nbsp;</p>
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        </div>
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      </div>
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<p><span style="font-family:arial,helvetica,sans-serif">5.Sequencing（Show&nbsp;results）</span></p>
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      <div class="section show-sec">
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        <nav class="navbar navbar-inverse">
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<p><span style="font-family:arial,helvetica,sans-serif">6.We&nbsp;then&nbsp;did&nbsp;alignment&nbsp;using&nbsp;BLAST.</span></p>
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          <div class="logo-container"><span class="logo">Flight iGEM</span></div>
+
 
−
          <h2>Online Collaboration<em></em></h2>
+
<p><img alt="" src="https://static.igem.org/mediawiki/2015/a/af/NEFU_China_E5ADCBCB-A89F-436F-AEBC-929E7CD914BD.png" style="height:461px; width:700px" /></p>
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        </nav>
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        <div class="container">
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<p><span style="font-family:arial,helvetica,sans-serif">&nbsp;The&nbsp;sequence&nbsp;was&nbsp;consist&nbsp;with&nbsp;that&nbsp;of&nbsp;<em>Salmonella&nbsp;enterica</em>&nbsp;subsp.&nbsp;<em>enterica&nbsp;serovar&nbsp;Typhimurium</em>&nbsp;str.&nbsp;LT2.</span></p>
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          <div class="row">
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            <div class="sub-container">
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<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:20px">B.&nbsp;Identification&nbsp;of&nbsp;Lactobacillus&nbsp;bulgaricus</span></span></p>
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              <div class="col-xs-6 col-md-6" style="text-align:center;"><img src="https://static.igem.org/mediawiki/2015/3/38/NEFU_China_93E048E2-4DF1-4B5C-B5A7-AAECD232B31F.png" /></div>
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<div class="col-xs-6 col-md-6 msg">
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<p><span style="font-family:arial,helvetica,sans-serif">1. PCR</span></p>
−
<p>A typical iGEM wiki can be divided into no less than 8 sections (like the home page, project, safety, team, and so on). Under each section, there are a lot of issues which you want to present. Just like the &lsquo;Project&rsquo; section, you may need to tell others about your background information, design, etc. Tedious as it is, we, the same as other 52 teams (52/56), believe that online collaboration can improve the efficiency of creating a wiki. Flight iGEM provides you an online platform, in which you can collaborate with your teammates, so every teammate can create page and edit them at any time or anywhere. To avoid version conflict(covered by others&rsquo; edition), we mimic the concept of SVN. You can click <a href="#" data-toggle="modal" data-backdrop="" data-target="#vcModal">me</a> to watch a short video which explains how it works.</p>
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−
</div>
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<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/6d/NEFU_China_D7858136-01CC-46B3-81F6-968BA3780EF0.png" style="width:700px" /></p>
−
            </div>
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−
          </div>
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<p style="text-align:center">&nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/b/b5/NEFU_China_3722D6C0-A70A-4DBB-B538-ED40DC3D889D.png" style="height:150px; width:100px" /></p>
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        </div>
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−
      </div>
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<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 3.&nbsp;1. Marker (DL2000) ; 2.&nbsp;<span style="font-size:14px"><em>Lactobacillus&nbsp;bulgaricus&nbsp;</em>16s rRNA</span></span></p>
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      <div class="section show-sec">
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        <nav class="navbar navbar-inverse">
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<p><span style="font-family:arial,helvetica,sans-serif">2.&nbsp;Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;see&nbsp;protocol)</span></p>
−
          <div class="logo-container"><span class="logo">Flight iGEM</span></div>
+
 
−
          <h2>Deadline alarm<em></em></h2>
+
<p><img alt="" src="https://static.igem.org/mediawiki/2015/d/d6/NEFU_China_1C19B503-1B40-4A7E-A648-1BCD39B79DAC.png" style="height:117px; width:700px" /></p>
−
        </nav>
+
 
−
        <div class="container">
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<p><span style="font-family:arial,helvetica,sans-serif">3.&nbsp;Transformation&nbsp;(<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a>)</span></p>
−
          <div class="row">
+
 
−
            <div class="sub-container">
+
<p><span style="font-family:arial,helvetica,sans-serif">4.&nbsp;Colony&nbsp;PCR</span></p>
−
                <div class="col-xs-12 col-md-12" style="text-align:center;"><img src="https://static.igem.org/mediawiki/2015/9/9b/NEFU_China_3702A597-6FFB-4715-990E-36F15066FBF3.png" /></div>
+
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/10/NEFU_China_A09473EB-D1C4-4E0E-84C1-166D737F2C94.png" style="height:399px; width:700px" /></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;<span style="color:rgb(0, 255, 255)"><span style="font-size:28px"><img alt="" src="https://static.igem.org/mediawiki/2015/6/6a/NEFU_China_087DB8ED-A25B-471E-903F-30722BDBA545.png" style="height:127px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig&nbsp;4.&nbsp;1. Marker (DL2000) ; 2.</span><span style="font-family:arial,helvetica,sans-serif">3.4.5.6&nbsp;</span><span style="font-family:arial,helvetica,sans-serif">each for one single colony; 7</span><span style="font-family:arial,helvetica,sans-serif">. Negative control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">5. Sequencing（Show&nbsp;results）</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">6. We&nbsp;then&nbsp;did&nbsp;alignment&nbsp;using&nbsp;BLAST.</span></p>
 +
 
 +
<p style="margin-left:18pt"><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequence&nbsp;was&nbsp;almost&nbsp;consist&nbsp;with&nbsp;that&nbsp;of&nbsp;<em>Lactobacillus&nbsp;delbrueckii</em>&nbsp;subsp.<em>&nbsp;Bulgaricus</em>&nbsp;str.&nbsp;Lb14.&nbsp;The&nbsp;differences&nbsp;were&nbsp;caused&nbsp;by&nbsp;subculturing&nbsp;perhaps.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:20px">Gene&nbsp;Cloning&nbsp;of<em>&nbsp;Lsr&nbsp;A,&nbsp;B,&nbsp;C,&nbsp;D,&nbsp;R&nbsp;</em>and&nbsp;<em>K</em></span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:16px">1.&nbsp;</span><span style="font-size:18px">Gene&nbsp;isolation&nbsp;using&nbsp;PCR</span>&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">(Restriction&nbsp;enzyme&nbsp;sites&nbsp;are&nbsp;indicated&nbsp;in&nbsp;red&nbsp;font)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><em>lsrA</em></span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/3c/NEFU_China_890BDFC2-9990-40D4-897D-CFA4E7E07181.png" style="height:353px; width:700px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><em>lsrB</em></span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/2/23/NEFU_China_E854482B-4DC9-40E3-8445-9FF5E79E7764.png" style="height:350px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><em><span style="font-size:18px">lsrC</span></em></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/1c/NEFU_China_084987BB-1EBE-415F-9049-FEFEA75EC092.png" style="height:344px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><em><span style="font-size:18px">lsrD</span></em></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/66/NEFU_China_B6FF9108-6231-4FE1-9032-6A754A410CC8.png" style="height:344px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><em>lsrK</em></span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/0/0b/NEFU_China_FD688B56-317E-43BD-8B4B-39766082AAB0.png" style="height:374px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><em>LsrR</em></span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/f4/NEFU_China_EE311814-A6B0-4227-ACFC-90A1272E3097.png" style="height:428px; width:700px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/1/1c/NEFU_China_9D7F3095-FE86-4E6D-853C-681D7A4B603F.jpg" style="height:253px; margin-left:350px; margin-right:350px; width:400px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 5.&nbsp;PCR&nbsp;products&nbsp;of&nbsp;lsrA,&nbsp;B&nbsp;and&nbsp;C&nbsp;&nbsp;1.Marker&nbsp;(DL2000) ;&nbsp;2.lsrA&nbsp;(1536bp) ;&nbsp;3.Negative&nbsp;control;&nbsp;4.lsrB&nbsp;(1023bp) ;&nbsp;5.Negative&nbsp;control;&nbsp;6.lsrC&nbsp;(1089bp) ;&nbsp;7.Negative&nbsp;control</span><img alt="" src="https://static.igem.org/mediawiki/2015/d/dd/NEFU_China_0954A0AA-5C06-49C4-A6EE-F63B36DE7E69.jpg" style="height:275px; margin-left:10px; margin-right:10px; width:400px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 6. PCR&nbsp;products&nbsp;of&nbsp;lsrD,&nbsp;R&nbsp;and&nbsp;K&nbsp;&nbsp;1.Marker&nbsp;(DL2000) ;&nbsp;2.lsrD&nbsp;(1002bp) ;&nbsp;3.Negative&nbsp;control;&nbsp;4.lsrR&nbsp;(960bp) ;&nbsp;5.Negative&nbsp;control;&nbsp;6.lsrK&nbsp;(1593bp) ;&nbsp;7.Negative&nbsp;control</span></p>
 +
 
 +
<p><span style="font-size:18px"><span style="font-family:arial,helvetica,sans-serif">2.Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;see&nbsp;protocol)</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_B2E2091E-C260-4EE2-949B-3EA94A5E2D1D.png" style="height:117px; width:700px" /><img alt="" src="https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_B2E2091E-C260-4EE2-949B-3EA94A5E2D1D.png" style="height:117px; width:700px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">3.Transformation&nbsp;(<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a>）</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">4.Colony&nbsp;PCR</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">lsrA</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><img alt="" src="https://static.igem.org/mediawiki/2015/d/d8/NEFU_China_9C5A87D7-9823-4A7F-A364-2BF5A0F9577B.png" style="height:389px; width:700px" /></span></span></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/b/ba/NEFU_China_B8CBC470-E785-4DF8-8F2E-DE83D315F6EB.jpg" style="height:277px; margin-left:350px; margin-right:350px; width:400px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 7.&nbsp;Clony&nbsp;PCR&nbsp;results.&nbsp;1.&nbsp;Marker&nbsp;(DL2000) ;&nbsp;2.3.4.5&nbsp;each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony (1536bp) ;&nbsp;6.Negative&nbsp;control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">lsrB</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><img alt="" src="https://static.igem.org/mediawiki/2015/3/3f/NEFU_China_D69BDDE6-652A-41F4-8DDA-7ACC82AAF452.png" style="width:700px" /></span></span></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/5/50/NEFU_China_5818DF91-9376-471B-8B85-7967C5F0F3B7.jpg" style="height:289px; margin-left:350px; margin-right:350px; width:400px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 8.&nbsp;Clony&nbsp;PCR&nbsp;results.&nbsp;1.&nbsp;Marker&nbsp;(DL2000) ;&nbsp;2.3.4&nbsp;each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony (1023) ;&nbsp;5.Negative&nbsp;control</span></p>
 +
 
 +
<p><strong><span style="font-size:18px">lsrC</span></strong></p>
 +
 
 +
<p><strong><span style="font-size:18px"><img alt="" src="https://static.igem.org/mediawiki/2015/6/6f/NEFU_China_BAC45DDC-FF3C-4A95-9076-4080D545E24D.png" style="height:428px; width:700px" /></span></strong></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/5/5e/NEFU_China_406D2C32-9C2C-479E-A080-414583438808.jpg" style="height:350px; margin-left:350px; margin-right:350px; width:350px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 9.&nbsp;Clony&nbsp;PCR&nbsp;results.&nbsp;1.&nbsp;Marker&nbsp;(DL2000) ;&nbsp;2.3.4.5&nbsp;each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony (1089bp) ;&nbsp;6.Negative&nbsp;control</span></p>
 +
 
 +
<p>sequencing</p>
 +
 
 +
<p><span style="font-size:18px">lsrD</span></p>
 +
 
 +
<p><span style="font-size:18px"><img alt="" src="https://static.igem.org/mediawiki/2015/e/eb/NEFU_China_3B850D6B-F363-4603-95DC-E802D5BB387B.png" style="width:700px" /></span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/8/86/NEFU_China_705C5ED8-2845-48E8-B4F2-574B24E8CD6B.jpg" style="height:343px; margin-left:350px; margin-right:350px; width:350px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 10.&nbsp;Clony&nbsp;PCR&nbsp;results.&nbsp;1.&nbsp;Marker&nbsp;(DL2000) ;&nbsp;2.3.4.5&nbsp;each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony (1002bp) ;&nbsp;6.Negative&nbsp;control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">lsrK</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><img alt="" src="https://static.igem.org/mediawiki/2015/7/77/NEFU_China_B80CF3CA-0BE7-4DAB-87D7-B77B87B361BA.png" style="width:700px" /></span></span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/5/59/NEFU_China_E346D693-FE67-4890-A9AB-86FAD9F8068D.jpg" style="height:350px; margin-left:350px; margin-right:350px; width:350px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 11.&nbsp;Clony&nbsp;PCR&nbsp;results.&nbsp;1.&nbsp;Marker&nbsp;(DL2000) ;&nbsp;2.3.4.5&nbsp;each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony(1593bp) ;&nbsp;6.Negative&nbsp;control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">lsrR</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><img alt="" src="https://static.igem.org/mediawiki/2015/c/ca/NEFU_China_4EE6A139-BDFC-4DBB-AF24-2C55019DDE48.png" style="width:700px" /></span></span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/8/83/NEFU_China_0E266A05-C035-4B64-A7F3-2A86DD198974.jpg" style="height:390px; margin-left:350px; margin-right:350px; width:350px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 12.&nbsp;Clony&nbsp;PCR&nbsp;results.&nbsp;1.&nbsp;Marker&nbsp;(DL2000) ;&nbsp;2.3.4.5&nbsp;each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony;&nbsp;6.Negative&nbsp;control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">5.sequencing</span></span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/8/85/NEFU_China_1D4FCA4D-2694-4D92-BAE4-D29C27A89915.png" style="height:370px; width:900px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 13.&nbsp;Sequencing&nbsp;result&nbsp;of&nbsp;LsrA</span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/c/c5/NEFU_China_6BD935D8-F97F-4490-81E5-6A5ECBB665FC.png" style="height:216px; width:900px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 14.&nbsp;Sequencing&nbsp;result&nbsp;of&nbsp;LsrB</span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/2/21/NEFU_China_82ED9097-982D-454B-98B5-AB52A5211EF1.png" style="height:311px; width:900px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 15.&nbsp;Sequencing&nbsp;result&nbsp;of&nbsp;LsrC</span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/d/df/NEFU_China_3C6BEB94-FDC3-410E-AEE9-676CD8312D95.png" style="height:278px; width:900px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 16.&nbsp;Sequencing&nbsp;result&nbsp;of&nbsp;LsrD</span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/8/80/NEFU_China_19EA3109-A08F-4BBD-8D6A-723289063F0E.png" style="height:415px; width:900px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 17.&nbsp;Sequencing&nbsp;result&nbsp;of&nbsp;LsrK</span></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/4/4f/NEFU_China_B61B8629-374E-4FC6-AF63-A2E44E7BA32F.png" style="height:277px; width:900px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 18.&nbsp;Sequencing&nbsp;result&nbsp;of&nbsp;LsrR</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">All&nbsp;the&nbsp;sequencing&nbsp;results&nbsp;were&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation,&nbsp;except&nbsp;for&nbsp;LsrD.&nbsp;There&nbsp;was&nbsp;a&nbsp;wrong&nbsp;base&nbsp;pair&nbsp;in&nbsp;the&nbsp;sequences&nbsp;we&nbsp;cloned,&nbsp;so&nbsp;we&nbsp;chose&nbsp;SOE(Splicing&nbsp;by&nbsp;overlap&nbsp;extension)-PCR&nbsp;to&nbsp;fix&nbsp;this&nbsp;error.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">A.&nbsp;Primary&nbsp;PCR&nbsp;reaction</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Segment1-lsrD1</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/d/da/NEFU_China_8B1F2BCB-3FB8-40F7-98C8-9173416417A8.png" style="height:411px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Segment2-lsrD2</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/0/08/NEFU_China_29EAEE3C-2E1A-41D5-BBA3-7293C61B8124.png" style="height:399px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;</span><img alt="" src="https://static.igem.org/mediawiki/2015/5/55/NEFU_China_D8939993-F487-49F1-B603-47E5C8A9E385.png" style="height:200px; width:200px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 19. LsrD1, LsrD2</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">B.&nbsp;Overlapping&nbsp;and&nbsp;elongation</span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/35/NEFU_China_291C996B-3652-4073-8F85-4351A3B8BCA0.png" style="height:307px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px">C.&nbsp;Second&nbsp;PCR&nbsp;reaction</span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/c/c0/NEFU_China_032E37C2-7F01-4D46-8AE5-18B072CF5100.png" style="height:386px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt"><span style="color:rgb(255, 215, 0)"><span style="font-family:arial,helvetica,sans-serif">图</span><span style="font-family:arial,helvetica,sans-serif">电泳</span></span></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="color:rgb(0, 0, 255)"><span style="font-family:arial,helvetica,sans-serif">Fig 20. 1. LsrD1,2.LsrD2,3.LsrD</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/b/bb/NEFU_China_8EF057C0-7F20-44BD-9362-D3CCBBF166BB.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;(see&nbsp;protocol)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Clone&nbsp;pcr&nbsp;</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/3b/NEFU_China_EB92ADF4-1C00-4F5F-BA7F-C0A4111A95C6.png" style="width:700px" /></p>
 +
 
 +
<p style="text-align:center"><img alt="" src="https://static.igem.org/mediawiki/2015/2/23/NEFU_China_ECFDFF16-BEA0-4F4C-8EF0-D80F6D7942D4.png" style="height:150px; width:200px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig&nbsp;21. 1. Marker&nbsp;(DL2000) ;&nbsp;2.3.4.5.each&nbsp;for&nbsp;one&nbsp;single&nbsp;colony(1002bp) ;&nbsp;6.Negative&nbsp;control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Sequencing</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/9/99/NEFU_China_E6C39C29-FC7F-43D2-ACD5-B1EC15673CD5.png" style="height:171px; width:552px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">As&nbsp;we&nbsp;see,&nbsp;the&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="margin-left:18pt"><img alt="" src="https://static.igem.org/mediawiki/2015/5/55/NEFU_China_D8939993-F487-49F1-B603-47E5C8A9E385.png" style="height:150px; width:150px" /></p>
 +
 
 +
<p>&nbsp;</p>
 +
<div>&nbsp;</div>
 +
 
 +
<div>
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong><span style="font-size:28px">Plasmid&nbsp;construction</span></strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">&nbsp;All&nbsp;designed&nbsp;fragments&nbsp;needed&nbsp;in&nbsp;plasmid&nbsp;construction&nbsp;were&nbsp;replicated&nbsp;using&nbsp;PCR&nbsp;.&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong><span style="font-size:18px">1.&nbsp;pNZ8148-lsrA</span></strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrA&nbsp;</strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pEASY-T5&nbsp;cloning&nbsp;vector&nbsp;with&nbsp;LsrA)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit（see&nbsp;protocol）</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/6d/NEFU_China_94713099-D0AB-4D58-8DA1-3E62FFA0D8B3.png" style="height:428px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/1b/NEFU_China_7A977166-D8B0-420B-9844-F0A58C8CB665.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/17/NEFU_China_39C0A08F-096B-48C1-9764-E5F5816EC6F4.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/2/20/NEFU_China_18B85C22-8EF6-4DD2-9E14-40D95C2ABE92.png" style="height:428px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/e/e7/NEFU_China_0ECC36ED-F12A-4C7E-A93B-FDF1212BEED6.png" style="height:225px; width:300px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp;&nbsp;Fig 22.1.&nbsp;Marker (DL2000) ; 2. Postive control (1536bp) ; 3.4.&nbsp;each for one single colony; 5. Negative control&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_B6CBC1C6-A178-4477-99AC-F9AC10532BCD.png" style="height:103px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<span style="color:rgb(255, 0, 0)">&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/1/1f/NEFU_China_D8A4081C-855E-4C56-BD09-F7D0AA3900F3.png" style="height:200px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig 23.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1536bp) ; 3. digest vector (3167bp) ; 4.pcr product (1536bp)</span></p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/b/b6/NEFU_China_B5E69330-87BC-4C0D-9F33-34658ACE5A93.png" style="height:350px; width:350px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><strong>2.&nbsp;pNZ8148-lsrB</strong></span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pEASY-T5&nbsp;cloning&nbsp;vector&nbsp;with&nbsp;LsrB)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit（see&nbsp;protocol）</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrB&nbsp;</strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/2/22/NEFU_China_24BACE72-9696-4E8F-996A-A39F77BCD376.png" style="width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/fb/NEFU_China_CDF8CF67-9BA9-4096-BF87-D80C0AE9C68A.png" style="width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/15/NEFU_China_BA1439C7-3254-4BE4-952D-BE481A8CD057.png" style="width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/d/d8/NEFU_China_3434BA7A-00BA-42E4-B1E8-1A13C4ACF4EA.png" style="height:429px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="color:rgb(255, 0, 0)"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/5/5c/NEFU_China_F40B0503-6325-44AF-8C6B-F191A326DCA3.png" style="height:250px; width:350px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp;Fig 24.&nbsp;1.&nbsp;Marker (DL2000) ; 2. Postive control (1023bp) ; 3.4.5.6.7.&nbsp;each for one single colony; 8. Negative control&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/d/df/NEFU_China_2C907F60-37C0-4A41-B0E1-EAE9AF7B66AB.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="text-align:center"><span style="color:rgb(255, 0, 0)"><img alt="" src="https://static.igem.org/mediawiki/2015/1/1f/NEFU_China_48721673-9D75-4F41-84D3-5C6E19FC3E5C.png" style="height:200px; width:150px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 25.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1023bp) ; 3. digest vector (3167bp) ; 4.pcr product (1023bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/e/e9/NEFU_China_31A684E7-E52D-4BA2-B06F-AAE167675891.png" style="height:350px; width:350px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:18px"><strong>3.&nbsp;pNZ8148-lsrC</strong></span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pEASY-T5&nbsp;cloning&nbsp;vector&nbsp;with&nbsp;LsrC)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit（&nbsp;see&nbsp;protocol）</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrC&nbsp;</strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/1a/NEFU_China_E23AD187-ABD7-4476-A15D-1CE8F6D341C3.png" style="height:379px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/5/5c/NEFU_China_FBA645D3-053B-45F5-BF6C-FB7D9CA02BA4.png" style="width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/7/7e/NEFU_China_EF7A8BD4-526E-4523-9CCB-3A647ADB2CAB.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/8/83/NEFU_China_ED5282F5-23BE-44D1-AB08-BE640545F7D2.png" style="height:428px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/7/76/NEFU_China_12A38364-2AD5-4C80-98D8-6461FDE0218A.png" style="height:156px; width:165px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 26.&nbsp;1. Marker (DL2000) ; 2. Postive control (1044bp) ; 3.4.5.6. each for one single colony; 7. Negative control</span>&nbsp;</p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/5/55/NEFU_China_941806DA-0880-4966-B717-B339ABD5E15F.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center">&nbsp;</p>
 +
 
 +
<p style="margin-left:18pt; text-align:center">&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span>&nbsp;&nbsp;&nbsp;&nbsp;<span style="color:rgb(255, 0, 0)">&nbsp;<span style="font-family:arial,helvetica,sans-serif">Fig 27.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1044bp)&nbsp;; 3. digest vector (3167bp) ; 4.pcr product (1044bp)</span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/f/fb/NEFU_China_9A99958D-EDE9-4849-A907-32867121C4FF.png" style="height:350px; width:350px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong><span style="font-size:18px">4.&nbsp;pNZ8148-lsrD</span></strong></span></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pEASY-T5&nbsp;cloning&nbsp;vector&nbsp;with&nbsp;LsrD)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrD&nbsp;</strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/7/72/NEFU_China_80F6A3BD-657A-497B-8AF4-970844664E23.png" style="height:379px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt"><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/b/b8/NEFU_China_23CDFBDF-A7D5-4660-AC6F-9AD84FB35942.png" style="height:157px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/d/da/NEFU_China_149D0F60-9342-4910-BA55-2680DC1E313B.png" style="height:119px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/4/47/NEFU_China_8794F247-02E3-4D97-8041-C9A4E7CEFDA4.png" style="height:428px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><img alt="" src="https://static.igem.org/mediawiki/2015/1/15/NEFU_China_967B6351-6F71-4272-92AA-04BF6EF14561.png" style="height:143px; width:220px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 28.&nbsp;1. Marker (DL2000) ; 2. Postive control (1000bp) ; 3.4.5.6. each for one single colony; 7. Negative control&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/3d/NEFU_China_3D3DE586-3ED2-4F91-830A-7DCB99A41AE8.png" style="height:86px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<span style="color:rgb(255, 0, 0)">&nbsp;&nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/9/91/NEFU_China_944D1AE5-D310-462B-8277-B98E73168B66.png" style="height:200px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig 29.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1002bp) ; 3. digest vector (3167bp) ; 4.pcr product (1002bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p style="margin-left:18pt"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:16px"><strong>sequencing</strong></span></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/6/6c/NEFU_China_881B8214-F510-43C2-91FA-CF5AC142C518.png" style="height:350px; width:350px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong><span style="font-size:18px">5.&nbsp;pNZ8148-lsrK</span></strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pEASY-T5&nbsp;cloning&nbsp;vector&nbsp;with&nbsp;LsrK)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrK</strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/37/NEFU_China_95C9EA81-6633-48CA-BE0F-0C0C3FAB062B.png" style="width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/d/d1/NEFU_China_288A9D71-88DA-425A-967D-D25D54FBC136.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/16/NEFU_China_D51B611E-A3F0-4DBF-B64D-B108934C81A9.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/4/45/NEFU_China_C99CD053-9661-4A10-8CAE-3840AB97E4A8.png" style="width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-size:24px"><span style="color:rgb(0, 255, 255)"><img alt="" src="https://static.igem.org/mediawiki/2015/2/28/NEFU_China_2293F963-4AF7-44BB-8BC9-1F38560C6973.png" style="height:200px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 30.&nbsp;1. Marker (DL2000) ; 2. Postive control (1593bp) ; 3.4.5. each for one single colony; 6. Negative control&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/7/7a/NEFU_China_E2E5B4B4-135E-4DA6-BD61-430962B5DDFD.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<span style="color:rgb(255, 0, 0)">&nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/0/0c/NEFU_China_F7463EFC-3ABE-4F33-9ECA-32054C0D9F58.png" style="height:300px; width:300px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;</span><br />
 +
<span style="font-family:arial,helvetica,sans-serif">Fig 31.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1593bp) ; 3. digest vector (3167bp) ; 4.pcr product (1593bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/a/ad/NEFU_China_44B8EC4F-CAEA-4DCD-84EF-843DDF9AA5B8.png" style="height:350px; width:350px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong><span style="font-size:18px">6.&nbsp;pNZ8148-lsrR</span></strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pEASY-T5&nbsp;cloning&nbsp;vector&nbsp;with&nbsp;LsrR)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrR</strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/a/a0/NEFU_China_96E3CC7B-17FD-4DD8-9847-242EB7BB5266.png" style="width:700px" /><span style="font-family:arial,helvetica,sans-serif">&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/60/NEFU_China_C393A81F-2AC5-40D6-A4FB-F47C14B6D3E6.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/63/NEFU_China_D95E46ED-88DC-4C03-AAB8-8013D7CC1C3B.png" style="height:111px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/b/bf/NEFU_China_8B5B6280-B089-4BBD-B00C-3164FE9A3BF9.png" style="width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="color:rgb(255, 0, 0)"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/8/8f/NEFU_China_49AC3B23-7060-47B0-99BA-CDA1E7955793.png" style="height:123px; width:169px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 32.&nbsp;1. Marker (DL2000) ; 2. Postive control (960bp) ; 3.4.5.6.7.each for one single colony; 8. Negative control&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/30/NEFU_China_2E868823-CAF5-46C2-982C-1B5DB3420370.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<span style="color:rgb(255, 0, 0)"><img alt="" src="https://static.igem.org/mediawiki/2015/5/58/NEFU_China_C8ED067B-D6B0-4AEB-9056-7F6286556087.png" style="height:300px; width:300px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><br />
 +
<span style="font-family:arial,helvetica,sans-serif">Fig 33.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,960bp) ; 3. digest vector (3167bp) ; 4.pcr product (960bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/b/b2/NEFU_China_6EF9C459-8175-4A86-A80A-D3FECB11A99A.png" style="height:350px; width:350px" /></span></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
  
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<p style="margin-left:18pt">&nbsp;</p>
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<em class="up"></em>
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<p>According to our investigation among 65 teams, 18 of them thought there were too many deadlines in iGEM and they might be forgotten easily. Moreover，43 teams surveyed indicated that it would be better if there are certain reminders for the deadlines. As we all know, iGEM is a competition composed of a lot of&nbsp;events. To help users manage their time better, Flight iGEM also covers a deadline reminder. Most noteworthy is that this reminder is able to convert time zones automatically according to the users&#39; geographic positions.</p>
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<p><strong><span style="font-size:16px"><span style="font-family:arial,helvetica,sans-serif">7.&nbsp;pHY300PLK-plsr-blue-T</span></span></strong></p>
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      <div class="section show-sec">
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        <nav class="navbar navbar-inverse">
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          <div class="logo-container"><span class="logo">Flight iGEM</span></div>
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          <h2>What's for the iGEM Server?<em></em></h2>
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        </nav>
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        <div class="container">
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                <div class="col-xs-6 col-md-6" style="text-align:center;"><img class="himg" src="https://static.igem.org/mediawiki/2015/d/d8/Queue.png" /></div>
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<div class="col-xs-6 col-md-6 msg">
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<p><span style="font-family:arial,helvetica,sans-serif">1.&nbsp;Molecular&nbsp;biology&nbsp;techniques:&nbsp;SOE&nbsp;(Splicing&nbsp;by&nbsp;overlap&nbsp;extension)&nbsp;PCR</span></p>
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<p>Considering that 83% teams (54/65) have encountered the situation that the&nbsp;iGEM server got slower or went down when it is close to the freeze of wiki, we decide to linearize&nbsp;all our request. It means if you want to build up a page and post it to iGEM server, you have to queue for a while until people who requested before you finished their query. To speed up all the requests, we create more than ten queues, which we believe is enough to handle them. And we hold the opinion that our platform will contribute a lot to ease the burden of iGEM servers, if it is accepted by other teams.</p>
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<h4 class="modal-title" id="vcModalLabel">How version control works</h4>
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<source src="https://static.igem.org/mediawiki/2015/4/49/NEFU_China_vc.ogg" type="video/ogg">
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    <!-- Include all compiled plugins (below), or include individual files as needed -->
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    $(document).ready(function() {
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      $('#fullpage').fullpage({
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        anchors: ['intro', 'builder', 'collaboration', 'deadline', '4igem'],
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        navigationTooltips: ['Some facts about creating a wiki', 'Flight iGEM Magic', 'Online Collaboration', 'Deadline', 'For iGEM']
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<p><span style="font-size:16px"><strong><span style="font-family:arial,helvetica,sans-serif">A.&nbsp;Primary&nbsp;PCR&nbsp;reaction</span></strong></span></p>
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<p><strong><span style="font-family:arial,helvetica,sans-serif">Segment1-plsr</span></strong></p>
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      var chart1 = {
+
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/f3/NEFU_China_31952388-2D19-4FA8-8DBD-63EDA1E2CB4F.png" style="height:344px; width:700px" /></p>
−
        labels : ['hard', 'tedious', 'not difficult', 'pleasant'],
+
−
        datasets : [
+
−
          {
+
−
            fillColor:"rgba(220,220,220,0.5)",
+
−
            strokeColor:"rgba(220,220,220,0.8)",
+
−
            highlightFill:"rgba(220,220,220,0.75)",
+
−
            highlightStroke:"rgba(220,220,220,1)",
+
−
            data: [24, 32, 6, 4],
+
−
          }
+
−
        ]
+
−
      }
+
−
     
+
  
−
      var ctx_1 = document.getElementById("slide-canvas-1").getContext("2d");
+
<p><strong><span style="font-family:arial,helvetica,sans-serif">Segment2-&nbsp;Blue-T</span></strong></p>
−
      window.myBar = new Chart(ctx_1).Bar(chart1, {responsive: true});
+
  
−
    });
+
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/f7/NEFU_China_B4879185-4C26-4FC5-A133-5F5056B51D02.png" style="height:399px; width:700px" /></p>
−
    </script>
+
 
−
   </body>
+
<p style="margin-left:18pt; text-align:center"><span style="color:rgb(255, 0, 0)"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/f/f9/NEFU_China_FAFBCF0A-D050-461A-A4A2-825CD2A6B4E2.png" style="height:150px; width:150px" /></span></span></p>
−
   </div>
+
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig&nbsp;34.&nbsp;1. Marker (DL2000) ; 2. Blue-T (878bp) ; 3. plsr (250bp)</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">B.&nbsp;Overlapping&nbsp;and&nbsp;elongation</span></strong></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/0/0b/NEFU_China_6F230DAC-C5D6-4BDB-88AA-02DA124DC03F.png" style="height:278px; width:700px" /></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">C.&nbsp;Second&nbsp;PCR&nbsp;reaction</span></strong></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/8/8d/NEFU_China_1E5EE16F-C0AB-4C36-85FB-F6082564BA8C.png" style="height:374px; width:700px" /></p>
 +
 
 +
<p><span style="color:rgb(255, 0, 0)"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/9/9b/NEFU_China_1759504E-CD10-448E-A49A-81693C20EF31.png" style="height:200px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 35.&nbsp;1. Marker (DL2000) ; 2. pLsr-Blue-T (1128bp) ; 3. pLsr (250bp) ; 4. Blue-T (878bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/7/7d/NEFU_China_28CC959C-9C24-4F25-81A6-A7BD6F9AF770.png" style="height:119px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/8/8a/NEFU_China_02420433-A28A-4085-AF17-0C250FF0A0ED.png" style="height:112px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/37/NEFU_China_6AFC9ED0-9BC4-48F2-BD1F-472ADF2A92F7.png" style="height:403px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="color:rgb(255, 0, 0)"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/6/67/NEFU_China_52E5F2E1-0F82-4B6D-91ED-87E7215CACF5.png" style="height:150px; width:200px" /></span></span></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 36.&nbsp;1. Marker (DL2000) ; 2.3.&nbsp;each for one single colony; 4. Negative control;&nbsp;5</span><span style="font-family:arial,helvetica,sans-serif">. Postive control (1128bp)</span></p>
 +
 
 +
<p style="margin-left:18pt"><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p style="margin-left:18pt"><img alt="" src="https://static.igem.org/mediawiki/2015/4/47/NEFU_China_BAAEBCCD-55D3-49DD-9F51-442791AEC61B.png" style="height:102px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="color:rgb(255, 0, 0)"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/2/22/NEFU_China_2529692B-0C96-4516-AF67-92A828B120CE.png" style="height:150px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig 37.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (4870bp,1128bp) ; 3. digest vector (4870bp) ; 4.pcr product (1128bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/2/2b/NEFU_China_C5CA77A9-97DD-41B4-BE9C-38150F1BADEA.png" style="height:576px; width:620px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/69/NEFU_China_7F00C6E0-AEBB-40AF-8613-46A0CEA146BB.png" style="height:574px; width:621px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/3e/NEFU_China_9041CE19-1736-4CA5-9E0F-7003F5DD06AB.png" style="height:573px; width:622px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/a/ad/NEFU_China_8D0E718D-241A-4FB4-8E21-B8F5AEFF7395.png" style="height:592px; width:604px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/e/e2/NEFU_China_D27300FE-620E-4866-A371-503C2E7E0A8B.png" style="height:582px; width:565px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/3d/NEFU_China_37A6BF4C-2E3E-480C-BA88-0B5A709C2453.png" style="height:579px; width:633px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/5/57/NEFU_China_F3A0DE33-A449-4102-8FCC-CAE417A0169C.png" style="height:576px; width:633px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/a/a6/NEFU_China_BE2AE6D3-0A32-45C6-952B-48F85CB8FC21.png" style="height:582px; width:555px" /></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/2/2e/NEFU_China_29B8210B-9815-475D-B667-7865812B822D.png" style="height:579px; width:638px" /></p>
 +
 
 +
<p><span style="font-size:20px"><strong><span style="font-family:arial,helvetica,sans-serif">8.&nbsp;pBBR1MCS-5-lsrA</span></strong></span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Insert&nbsp;lsrA&nbsp;</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pNZ8148&nbsp;with&nbsp;LsrA)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></p>
 +
 
 +
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/c/c2/NEFU_China_93E23CE5-9BC9-46AA-8B81-EB60A6154CBA.png" style="height:348px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/f9/NEFU_China_DBC8232D-3C4F-45A1-B117-880DD13B520E.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/e/e1/NEFU_China_34C18741-B024-438E-A27C-68B7A2495375.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/fe/NEFU_China_11E19294-D9B6-46C5-8CCD-09991F733E28.png" style="height:427px; width:700px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/8/83/NEFU_China_325A7E57-B20E-4848-838D-9BFD4B801241.png" style="height:250px; width:300px" /></span></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 38.&nbsp;1. Marker (DL2000) ; 2. Postive control (1952bp)&nbsp;; 3.4.5.6.7. each for one single colony; 8. Negative control&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/c/cf/NEFU_China_8D7F0BEE-3055-456B-9200-6513B3EF7E05.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/8/80/NEFU_China_240B221E-02C5-4D9A-AA79-6CB566ED3546.png" style="height:170px; width:200px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span>&nbsp;&nbsp;&nbsp;<span style="font-family:arial,helvetica,sans-serif">&nbsp;Fig 39.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (4707bp,1952bp) ; 3. digest vector (4707bp) ; 4.pcr product (1952bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-size:20px"><strong><span style="font-family:arial,helvetica,sans-serif">9.&nbsp;pBBR1MCS-5-lsrC</span></strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pNZ8148&nbsp;with&nbsp;LsrC)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Insert&nbsp;lsrC&nbsp;</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/19/NEFU_China_67956AB6-06C7-4B1D-813A-C8C06B27DB44.png" style="height:428px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/1b/NEFU_China_F2FDDE95-1E32-4CDB-9271-A6446D7B41C4.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/f/fc/NEFU_China_401376A0-12F4-4A49-B3D9-DD005FC17FA8.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/7/7f/NEFU_China_E94743DF-F79B-4D8E-BB03-E25EC77F9AF8.png" style="height:420px; width:700px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-size:28px"><span style="color:rgb(0, 255, 255)"><img alt="" src="https://static.igem.org/mediawiki/2015/7/7c/NEFU_China_61F8EE29-8DAA-4232-8A60-C3F36DAECF37.png" style="height:200px; width:200px" /></span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 40.&nbsp;1. Marker (DL2000) ; 2. Postive control (1503bp) ; 3.4.5.6. each for one single colony; 7. Negative control</span>&nbsp;</p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/64/NEFU_China_95207A9B-71D0-414F-92F3-B318A86DA925.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt; text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/b/be/NEFU_China_3089B91F-DA4F-4575-99EC-6894AA28BD32.png" style="height:300px; width:300px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig 41.&nbsp;1. Marker (DL10000) ; 2. Double enmyze digestion (4707bp,1503bp) ; 3. digest vector (4707bp) ; 4.pcr product (1503bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;result&nbsp;is&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
 
 +
<p><span style="font-size:20px"><strong><span style="font-family:arial,helvetica,sans-serif">10.&nbsp;pBBR1MCS-5-lsrD</span></strong></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pNZ8148&nbsp;with&nbsp;LsrD)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><strong>Insert&nbsp;lsrD</strong>&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">1.PCR</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/9/9d/NEFU_China_F478B484-1697-46AB-BE81-40B946C8C959.png" style="height:428px; width:700px" /></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Double&nbsp;digestion&nbsp;(Takara)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/2/2b/NEFU_China_705F260A-117F-48E7-B075-D788F8C18E78.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Ligation&nbsp;(TaKaRa&nbsp;DNA&nbsp;Ligation&nbsp;Kit&nbsp;Ver.2.1<a href="http://www.takara.com.cn/?action=Page&amp;Plat=pdetail&amp;newsid=101&amp;subclass=1">see&nbsp;manual</a>)</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/e/eb/NEFU_China_033F7B8F-50B3-460B-89FA-1A05F4AE9F53.png" style="height:110px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;<a href="https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf">see&nbsp;protocol</a></span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/0/0a/NEFU_China_DB561A91-6641-470A-A160-6396F52823A8.png" style="height:420px; width:700px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/e/ef/NEFU_China_26803078-6A16-4F32-BA69-60A8E49B49BF.png" style="height:150px; width:150px" /><span style="color:rgb(255, 0, 0)">在该</span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 42.&nbsp;1. Marker (DL2000) ;&nbsp;2.3.4.5&nbsp;each for one single colony (1439bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Two-step&nbsp;enzyme&nbsp;digestion(Takara)&nbsp;for&nbsp;detection</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/7/75/NEFU_China_566EB48F-EDC1-410B-B218-AEB3CC949CCC.png" style="height:81px; width:700px" /></p>
 +
 
 +
<p style="margin-left:18pt"><span style="color:rgb(255, 0, 0)">&nbsp;<span style="font-size:36px">&nbsp;&nbsp; &nbsp; &nbsp;</span></span><span style="font-size:36px"><span style="color:rgb(0, 255, 255)">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;</span></span><img alt="" src="https://static.igem.org/mediawiki/2015/6/6a/NEFU_China_40E915B9-202B-4ACC-BC28-7A308B5A28CF.png" style="height:275px; width:250px" /><span style="font-size:36px"><span style="color:rgb(0, 255, 255)">&nbsp;</span></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig 43.&nbsp;1. Marker (DL10000) ; 2.&nbsp;</span><span style="font-family:arial,helvetica,sans-serif">digest vector (4707bp);</span><span style="font-family:arial,helvetica,sans-serif">&nbsp;</span><span style="font-family:arial,helvetica,sans-serif">3.&nbsp;</span><span style="font-family:arial,helvetica,sans-serif">Double enmyze digestion (4707bp,1439</span><span style="font-family:arial,helvetica,sans-serif">bp) ;</span><span style="font-family:arial,helvetica,sans-serif">&nbsp;4.pcr product (139</span><span style="font-family:arial,helvetica,sans-serif">bp)</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">The&nbsp;sequencing&nbsp;results are&nbsp;consistent&nbsp;with&nbsp;our&nbsp;designation.</span></p>
 +
<p><strong><span style="font-size:20px"><span style="font-family:arial,helvetica,sans-serif">Transformation&nbsp;in&nbsp;<em>Lactobacillus</em></span></span></strong></p>
 +
 
 +
<p><strong><span style="font-size:18px"><span style="font-family:arial,helvetica,sans-serif">Electrotransformation&nbsp;of&nbsp;LsrBRK</span></span></strong></p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Miniprep&nbsp;(pNZ8148&ndash;<em>LsrB</em>,&nbsp;pNZ8148&ndash;<em>LsrR</em>,&nbsp;pNZ8148&ndash;<em>LsrK</em>)&nbsp;with&nbsp;TIANprep&nbsp;Mini&nbsp;Plasmid&nbsp;Kit</span></strong></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">1.Preparation&nbsp;of&nbsp;competent&nbsp;cells&nbsp;</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">100&nbsp;&mu;l&nbsp;bacterial&nbsp;culture&nbsp;were&nbsp;inoculated&nbsp;into&nbsp;50ml&nbsp;of&nbsp;MRS&nbsp;and&nbsp;incubated&nbsp;at&nbsp;37&deg;C.&nbsp;After&nbsp;overnight&nbsp;growth,&nbsp;a&nbsp;culture&nbsp;at&nbsp;the&nbsp;beginning&nbsp;of&nbsp;the&nbsp;stationary&nbsp;phase&nbsp;was&nbsp;harvested&nbsp;by&nbsp;centrifugation.&nbsp;The&nbsp;bacteria&nbsp;were&nbsp;washed&nbsp;three&nbsp;times&nbsp;with&nbsp;cold&nbsp;electroporation&nbsp;buffer&nbsp;(PB).The&nbsp;cells&nbsp;were&nbsp;then&nbsp;resuspended&nbsp;in&nbsp;PB&nbsp;to&nbsp;an&nbsp;OD600&nbsp;of&nbsp;about&nbsp;50.</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">2.Electrotransfection&nbsp;</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">100&nbsp;&nbsp;&mu;l&nbsp;of&nbsp;the&nbsp;cell&nbsp;suspension&nbsp;was&nbsp;mixed&nbsp;with&nbsp;10&nbsp;&mu;l&nbsp;plasmid&nbsp;DNA(pNZ8148&ndash;<em>LsrB</em>,&nbsp;pNZ8148&ndash;<em>LsrR</em>,&nbsp;pNZ8148&ndash;<em>LsrK</em>).&nbsp;The&nbsp;sample&nbsp;was&nbsp;subjected&nbsp;to&nbsp;a&nbsp;2.4kV,&nbsp;200&Omega;,&nbsp;25&mu;F&nbsp;electric&nbsp;pulse&nbsp;in&nbsp;a&nbsp;0.2cm&nbsp;cuvette.</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">3.Recovery&nbsp;of&nbsp;transformants</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">950&nbsp;&mu;l&nbsp;SMRS&nbsp;was&nbsp;immediately&nbsp;added,&nbsp;and&nbsp;the&nbsp;cells&nbsp;were&nbsp;incubated&nbsp;for&nbsp;2&nbsp;h&nbsp;at&nbsp;37&deg;C&nbsp;before&nbsp;they&nbsp;were&nbsp;plated&nbsp;on&nbsp;MRS&nbsp;supplemented&nbsp;with&nbsp;the&nbsp;appropriate&nbsp;antibiotic.&nbsp;The&nbsp;plates&nbsp;were&nbsp;incubated&nbsp;at&nbsp;37&deg;C&nbsp;for&nbsp;2&nbsp;to&nbsp;3&nbsp;days&nbsp;under&nbsp;anaerobic&nbsp;conditions.</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">4.Clone&nbsp;identify</span></strong></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/e/e6/NEFU_China_2A32A9E4-41BC-4EF5-A396-74EA013FE6B9.png" style="height:592px; width:700px" /></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">We&nbsp;repeated&nbsp;this&nbsp;transformation&nbsp;process&nbsp;for&nbsp;several&nbsp;times.&nbsp;Unfortunatelly,&nbsp;not&nbsp;a&nbsp;single&nbsp;colony&nbsp;was&nbsp;grown&nbsp;the&nbsp;plate.</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Then&nbsp;we&nbsp;changed&nbsp;the&nbsp;parameters&nbsp;of&nbsp;electric&nbsp;pulse&nbsp;and&nbsp;tried&nbsp;several&nbsp;recovery&nbsp;medium,&nbsp;but&nbsp;we&nbsp;just&nbsp;got&nbsp;some&nbsp;unsatisfactory&nbsp;results.&nbsp;</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/a/ac/NEFU_China_F3EE6719-D8A6-4F55-B1CF-CC82059C567C.png" style="height:300px; width:300px" /></span></p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 44.&nbsp;1. Marker (DL2000)&nbsp;; 2.3.4.5.6.7 each for one single LsrB colony; 8.9.10.11.12.13 each for one single LsrR&nbsp;colony;</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">1. Marker (DL2000)&nbsp;; 2.3.4.5.6.7 each for one single LsrKcolony&nbsp;</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/f/f0/NEFU_China_A6318DC8-2957-4C64-9061-6686F9255552.png" style="height:300px; width:300px" /></span></p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 45.&nbsp;1. Marker (DL2000)&nbsp;; 2.3.4.5.6.7 each for one single LsrB colony; 8.9.10.11.12.13 each for one single LsrR&nbsp;colony;</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">1. Marker (DL2000)&nbsp;; 2.3.4.5.6.7 each for one single LsrKcolony&nbsp;</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/a/ad/NEFU_China_0C3ED3F4-8E71-4A6A-94CA-77035E009229.png" style="height:400px; width:400px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 46.&nbsp;1. Marker (DL10000)&nbsp;; 2.3.4.5.6. each for one single LsrB colony; 7.8.9.&nbsp;each for one single LsrR&nbsp;colony;</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;1. Marker (DL10</span><span style="font-family:arial,helvetica,sans-serif">000)&nbsp;; 2.3. each for one single LsrR&nbsp;colony; 4.5.6.7.</span><span style="font-family:arial,helvetica,sans-serif">8.</span><span style="font-family:arial,helvetica,sans-serif">each for one single Ls</span><span style="font-family:arial,helvetica,sans-serif">rk</span><span style="font-family:arial,helvetica,sans-serif">&nbsp;colony</span><span style="font-family:arial,helvetica,sans-serif">;&nbsp;</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Finally,&nbsp;we&nbsp;realized&nbsp;it&nbsp;was&nbsp;the&nbsp;concentration&nbsp;of&nbsp;the&nbsp;plasmids&nbsp;which&nbsp;led&nbsp;to&nbsp;the&nbsp;low&nbsp;efficiency&nbsp;in&nbsp;transformation.&nbsp;We&nbsp;then&nbsp;did&nbsp;plasmid&nbsp;Maxiprep&nbsp;(see&nbsp;protocol)&nbsp;and&nbsp;concentration,&nbsp;and&nbsp;obtained&nbsp;plasmids&nbsp;in&nbsp;a&nbsp;concentraion&nbsp;of&nbsp;about&nbsp;3000ng/&mu;L.&nbsp;We&nbsp;also&nbsp;added&nbsp;100&mu;L&nbsp;denatured&nbsp;salmon&nbsp;sperm&nbsp;DNA&nbsp;to&nbsp;the&nbsp;cuvette&nbsp;before&nbsp;electroporation.&nbsp;This&nbsp;time,&nbsp;we&nbsp;eventually&nbsp;gained&nbsp;positive&nbsp;colonies.</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Clone&nbsp;identify</span></strong></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/a/a0/NEFU_China_85EBC823-DCA7-402D-9EAF-19C3ABD712A3.png" style="height:450px; width:450px" /></span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 47.&nbsp;1. Marker (DL4000)&nbsp;; 2.3.4.5.6. each for one single LsrB colony; 7. Postive control (1023bp) ; 8.9.10.11.12.&nbsp;each for one single colony&nbsp;;&nbsp;13</span><span style="font-family:arial,helvetica,sans-serif">. Postive control (1593bp)</span></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;1. Marker (DL4000)&nbsp;; 2.3.4.5.6. each for one single LsrR&nbsp;colony; 7</span><span style="font-family:arial,helvetica,sans-serif">. Postive control (960bp);</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Electrotransformation&nbsp;of&nbsp;<em>plsr+blue&nbsp;</em>pigment+T</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Maxiprep&nbsp;(see&nbsp;protocol)&nbsp;and&nbsp;concentration&nbsp;of&nbsp;pHY300PLK-pLsr-&nbsp;amilCP。</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Clone&nbsp;identify</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/1/12/NEFU_China_25F1499D-4D64-42D5-8B98-EA16935BE2C3.png" style="height:420px; width:700px" /></p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/0/03/NEFU_China_4E03A65D-FD94-4FCD-9E63-5EE7937F8FE5.png" style="height:200px; width:200px" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig 48.1. Marker (DL2000) ; 2. Postive control (1128bp) ; 3.4. each for one single colony; 5. Negative control</span></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">&nbsp;The&nbsp;transformation&nbsp;procedures&nbsp;are&nbsp;similar&nbsp;to&nbsp;Electrotransformation&nbsp;of&nbsp;BRK</span></p>
 +
 
 +
<p><strong><span style="font-family:arial,helvetica,sans-serif">Clone&nbsp;identify</span></strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif">Colony&nbsp;PCR</span></p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
 +
 
 +
<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<img alt="" src="https://static.igem.org/mediawiki/2015/7/7d/NEFU_China_5F2D24C3-D76A-46F5-9A59-C1BCAB3A2271.png" /></p>
 +
 
 +
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig.？: 1. Marker (DL2000) ; 2.3.4.5.6. each for one single colony (486bp)&nbsp;; 7. Postive&nbsp;control</span>&nbsp;</p>
 +
 
 +
<p style="text-align:center">&nbsp;</p>
 +
 
 +
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Revision as of 14:43, 18 September 2015

Monthly


January
Our junior and senior students were attracted by the recruiting announcement for the iGEM 2015 in our university. After several interviews and competitive selections, our team was basically created.
February
Our team was growing. We communicated with previous iGEMers in our school to get more insights into iGEM. Meanwhile, we started to watch successful iGEM cases of previous years.
March
We wandered among wikis and gradually realized what the basic factors are for a good project. Brainstorms and weekly meet-ups promoted us to discover the quintessence of iGEM.
April
We urged ourselves to get deeper insights into those previous cases to catch their inspiration. After meet-ups with teammates and our instructors, a draft idea of a yogurt guarder came to our mind. To make sure our project was worth proceeding, we did a survey to find out how much attention people would pay to the quality of yogurt. Fortunately, we got the expected results, although we knew we still had a long way to go.
May
We primarily designed the basic structure of our project. Then we read numerous papers to make an integrated protocol of our lab work.Besides, we visited a dairy factory to learn the microbiological detection methods used in practical production of yogurt. We discovered that our method to be developed should have advantages over the traditional methods, especially in portability. 
In addition, it was essential for us to adapt to the wet lab work. We started with laboratory safety training. With the instruction of our instructors and previous iGEMers, we learned a lot of techniques in molecular biology.
June
With efforts, our wet lab work went smoothly.
July
This month, we organized public activity to popularize the knowledge of yogurt production and its quality inspection, and remind people to store yogurt properly. 
In addition, we did a survey to confirm the key factors which will attract customers’ attention if our yogurt guarder is developed into a real product. 
August
On 13th-15th, we attended the summit of Central China iGEM Consortium held by Peking University. We shared our experience with other teams and really learned a lot from other teams. Here we want to express our sincere thanks to them! 
Meanwhile, our lab works still proceeded. We encountered difficulty in the electrotransformation of bacteria, which delayed our project but was eventually resolved after multiple attempts.
September
Time flies. We seized the time to make everything done and put great efforts to the wiki building and preparation for the Giant Jamboree.

Labnote

Isolation of genomic DNA from bacteria             The genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 and Lactobacillus delbrueckii subsp. Bulgaricus str. Lb14 were isolated using the TIANamp Bacteria DNA Kit (see protocol). The concentration of genomic DNA was measured with a NanoDrop spectrophotometer.         

Strain identification

We applied 16S ribosomal DNA identification to identify the strain of Salmonella and Lactobacillus bought from a biotech company. Universal primers were designed according to the conserved sequences.

A. Identification of Salmonella typhimurium

1.PCR

                                                                 

                Fig 1. 1. Marker (2000bp DNA Ladder) ; 2. Salmonella typhimurium 16s rRNA

2. Ligation (TaKaRa DNA Ligation Kit see protocol)

3. Transformation (see protocol)

4. Colony PCR

Fig 2. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony; 6. Negative control 

5.Sequencing（Show results）

6.We then did alignment using BLAST.

 The sequence was consist with that of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2.

B. Identification of Lactobacillus bulgaricus

1. PCR

  

Fig 3. 1. Marker (DL2000) ; 2. Lactobacillus bulgaricus 16s rRNA

2. Ligation (TaKaRa DNA Ligation Kit see protocol)

3. Transformation (see protocol)

4. Colony PCR

                                                          

Fig 4. 1. Marker (DL2000) ; 2.3.4.5.6 each for one single colony; 7. Negative control

5. Sequencing（Show results）

6. We then did alignment using BLAST.

The sequence was almost consist with that of Lactobacillus delbrueckii subsp. Bulgaricus str. Lb14. The differences were caused by subculturing perhaps.

Gene Cloning of Lsr A, B, C, D, R and K

1. Gene isolation using PCR 

(Restriction enzyme sites are indicated in red font)

lsrA

 

lsrB

lsrC

lsrD

lsrK

LsrR

 

          

Fig 5. PCR products of lsrA, B and C  1.Marker (DL2000) ; 2.lsrA (1536bp) ; 3.Negative control; 4.lsrB (1023bp) ; 5.Negative control; 6.lsrC (1089bp) ; 7.Negative control

 

Fig 6. PCR products of lsrD, R and K  1.Marker (DL2000) ; 2.lsrD (1002bp) ; 3.Negative control; 4.lsrR (960bp) ; 5.Negative control; 6.lsrK (1593bp) ; 7.Negative control

2.Ligation (TaKaRa DNA Ligation Kit see protocol)

3.Transformation (see protocol）

4.Colony PCR

lsrA

 

            

Fig 7. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1536bp) ; 6.Negative control

lsrB

 

        

Fig 8. Clony PCR results. 1. Marker (DL2000) ; 2.3.4 each for one single colony (1023) ; 5.Negative control

lsrC

                    

Fig 9. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1089bp) ; 6.Negative control

sequencing

lsrD

                    

 

Fig 10. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1002bp) ; 6.Negative control

lsrK

                     

 

Fig 11. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony(1593bp) ; 6.Negative control

lsrR

            

Fig 12. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony; 6.Negative control

5.sequencing

                           

Fig 13. Sequencing result of LsrA

                           

Fig 14. Sequencing result of LsrB

                          

Fig 15. Sequencing result of LsrC

                          

Fig 16. Sequencing result of LsrD

                          

Fig 17. Sequencing result of LsrK

                          

Fig 18. Sequencing result of LsrR

All the sequencing results were consistent with our designation, except for LsrD. There was a wrong base pair in the sequences we cloned, so we chose SOE(Splicing by overlap extension)-PCR to fix this error.

A. Primary PCR reaction

Segment1-lsrD1

Segment2-lsrD2

                                                                                                                                          

Fig 19. LsrD1, LsrD2

B. Overlapping and elongation

C. Second PCR reaction

图电泳

Fig 20. 1. LsrD1,2.LsrD2,3.LsrD

Ligation

Transformation (see protocol)

Clone pcr 

 

Fig 21. 1. Marker (DL2000) ; 2.3.4.5.each for one single colony(1002bp) ; 6.Negative control

Sequencing

As we see, the sequencing result is consistent with our designation.

 

 

 

 

 

 

Plasmid construction

 All designed fragments needed in plasmid construction were replicated using PCR . 

1. pNZ8148-lsrA

Insert lsrA 

Miniprep (pEASY-T5 cloning vector with LsrA) with TIANprep Mini Plasmid Kit（see protocol）

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

                                                 

    Fig 22.1. Marker (DL2000) ; 2. Postive control (1536bp) ; 3.4. each for one single colony; 5. Negative control 

Two-step enzyme digestion(Takara) for detection

                                                                                                                                            

                  Fig 23. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1536bp) ; 3. digest vector (3167bp) ; 4.pcr product (1536bp)

 

The sequencing result is consistent with our designation.

2. pNZ8148-lsrB

Miniprep (pEASY-T5 cloning vector with LsrB) with TIANprep Mini Plasmid Kit（see protocol）

Insert lsrB 

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

   Fig 24. 1. Marker (DL2000) ; 2. Postive control (1023bp) ; 3.4.5.6.7. each for one single colony; 8. Negative control 

Two-step enzyme digestion(Takara) for detection

Fig 25. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1023bp) ; 3. digest vector (3167bp) ; 4.pcr product (1023bp)

The sequencing result is consistent with our designation.

3. pNZ8148-lsrC

Miniprep (pEASY-T5 cloning vector with LsrC) with TIANprep Mini Plasmid Kit（ see protocol）

Insert lsrC 

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

  

Fig 26. 1. Marker (DL2000) ; 2. Postive control (1044bp) ; 3.4.5.6. each for one single colony; 7. Negative control 

Two-step enzyme digestion(Takara) for detection

 

 

                     Fig 27. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1044bp) ; 3. digest vector (3167bp) ; 4.pcr product (1044bp)

The sequencing result is consistent with our designation.

4. pNZ8148-lsrD

 

Miniprep (pEASY-T5 cloning vector with LsrD) with TIANprep Mini Plasmid Kit

Insert lsrD 

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

Fig 28. 1. Marker (DL2000) ; 2. Postive control (1000bp) ; 3.4.5.6. each for one single colony; 7. Negative control 

Two-step enzyme digestion(Takara) for detection

           

                      Fig 29. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1002bp) ; 3. digest vector (3167bp) ; 4.pcr product (1002bp)

The sequencing result is consistent with our designation.

sequencing

5. pNZ8148-lsrK

Miniprep (pEASY-T5 cloning vector with LsrK) with TIANprep Mini Plasmid Kit

Insert lsrK

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

Fig 30. 1. Marker (DL2000) ; 2. Postive control (1593bp) ; 3.4.5. each for one single colony; 6. Negative control 

Two-step enzyme digestion(Takara) for detection

                   

                               
Fig 31. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1593bp) ; 3. digest vector (3167bp) ; 4.pcr product (1593bp)

The sequencing result is consistent with our designation.

6. pNZ8148-lsrR

Miniprep (pEASY-T5 cloning vector with LsrR) with TIANprep Mini Plasmid Kit

Insert lsrR

1.PCR

 

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

Fig 32. 1. Marker (DL2000) ; 2. Postive control (960bp) ; 3.4.5.6.7.each for one single colony; 8. Negative control 

Two-step enzyme digestion(Takara) for detection

          

                     
Fig 33. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,960bp) ; 3. digest vector (3167bp) ; 4.pcr product (960bp)

The sequencing result is consistent with our designation.

 

 

 

 

7. pHY300PLK-plsr-blue-T

1. Molecular biology techniques: SOE (Splicing by overlap extension) PCR

A. Primary PCR reaction

Segment1-plsr

Segment2- Blue-T

Fig 34. 1. Marker (DL2000) ; 2. Blue-T (878bp) ; 3. plsr (250bp)

B. Overlapping and elongation

C. Second PCR reaction

                                                                                                                                            

Fig 35. 1. Marker (DL2000) ; 2. pLsr-Blue-T (1128bp) ; 3. pLsr (250bp) ; 4. Blue-T (878bp)

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

Fig 36. 1. Marker (DL2000) ; 2.3. each for one single colony; 4. Negative control; 5. Postive control (1128bp)

Two-step enzyme digestion(Takara) for detection

                  Fig 37. 1. Marker (DL10000) ; 2. Double enmyze digestion (4870bp,1128bp) ; 3. digest vector (4870bp) ; 4.pcr product (1128bp)

The sequencing result is consistent with our designation.

8. pBBR1MCS-5-lsrA

Insert lsrA 

Miniprep (pNZ8148 with LsrA) with TIANprep Mini Plasmid Kit

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

 

Fig 38. 1. Marker (DL2000) ; 2. Postive control (1952bp) ; 3.4.5.6.7. each for one single colony; 8. Negative control 

Two-step enzyme digestion(Takara) for detection

                  Fig 39. 1. Marker (DL10000) ; 2. Double enmyze digestion (4707bp,1952bp) ; 3. digest vector (4707bp) ; 4.pcr product (1952bp)

The sequencing result is consistent with our designation.

9. pBBR1MCS-5-lsrC

Miniprep (pNZ8148 with LsrC) with TIANprep Mini Plasmid Kit

Insert lsrC 

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

Fig 40. 1. Marker (DL2000) ; 2. Postive control (1503bp) ; 3.4.5.6. each for one single colony; 7. Negative control 

Two-step enzyme digestion(Takara) for detection

                     Fig 41. 1. Marker (DL10000) ; 2. Double enmyze digestion (4707bp,1503bp) ; 3. digest vector (4707bp) ; 4.pcr product (1503bp)

The sequencing result is consistent with our designation.

10. pBBR1MCS-5-lsrD

Miniprep (pNZ8148 with LsrD) with TIANprep Mini Plasmid Kit

Insert lsrD 

1.PCR

Double digestion (Takara)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Transformation see protocol

Colony PCR

在该

Fig 42. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1439bp)

Two-step enzyme digestion(Takara) for detection

                     

                    Fig 43. 1. Marker (DL10000) ; 2. digest vector (4707bp); 3. Double enmyze digestion (4707bp,1439bp) ; 4.pcr product (139bp)

The sequencing results are consistent with our designation.

Transformation in Lactobacillus

Electrotransformation of LsrBRK

 

Miniprep (pNZ8148–LsrB, pNZ8148–LsrR, pNZ8148–LsrK) with TIANprep Mini Plasmid Kit

1.Preparation of competent cells 

100 μl bacterial culture were inoculated into 50ml of MRS and incubated at 37°C. After overnight growth, a culture at the beginning of the stationary phase was harvested by centrifugation. The bacteria were washed three times with cold electroporation buffer (PB).The cells were then resuspended in PB to an OD600 of about 50.

2.Electrotransfection 

100  μl of the cell suspension was mixed with 10 μl plasmid DNA(pNZ8148–LsrB, pNZ8148–LsrR, pNZ8148–LsrK). The sample was subjected to a 2.4kV, 200Ω, 25μF electric pulse in a 0.2cm cuvette.

3.Recovery of transformants

950 μl SMRS was immediately added, and the cells were incubated for 2 h at 37°C before they were plated on MRS supplemented with the appropriate antibiotic. The plates were incubated at 37°C for 2 to 3 days under anaerobic conditions.

4.Clone identify

We repeated this transformation process for several times. Unfortunatelly, not a single colony was grown the plate.

Then we changed the parameters of electric pulse and tried several recovery medium, but we just got some unsatisfactory results. 

 

Fig 44. 1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrB colony; 8.9.10.11.12.13 each for one single LsrR colony;

1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrKcolony 

           

 

Fig 45. 1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrB colony; 8.9.10.11.12.13 each for one single LsrR colony;

1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrKcolony 

Fig 46. 1. Marker (DL10000) ; 2.3.4.5.6. each for one single LsrB colony; 7.8.9. each for one single LsrR colony;

         1. Marker (DL10000) ; 2.3. each for one single LsrR colony; 4.5.6.7.8.each for one single Lsrk colony; 

Finally, we realized it was the concentration of the plasmids which led to the low efficiency in transformation. We then did plasmid Maxiprep (see protocol) and concentration, and obtained plasmids in a concentraion of about 3000ng/μL. We also added 100μL denatured salmon sperm DNA to the cuvette before electroporation. This time, we eventually gained positive colonies.

Clone identify

Fig 47. 1. Marker (DL4000) ; 2.3.4.5.6. each for one single LsrB colony; 7. Postive control (1023bp) ; 8.9.10.11.12. each for one single colony ; 13. Postive control (1593bp)

           1. Marker (DL4000) ; 2.3.4.5.6. each for one single LsrR colony; 7. Postive control (960bp);

Electrotransformation of plsr+blue pigment+T

Maxiprep (see protocol) and concentration of pHY300PLK-pLsr- amilCP。

Clone identify

Colony PCR

                                                                           

Fig 48.1. Marker (DL2000) ; 2. Postive control (1128bp) ; 3.4. each for one single colony; 5. Negative control

 The transformation procedures are similar to Electrotransformation of BRK

Clone identify

Colony PCR

 

                                                              

Fig.？: 1. Marker (DL2000) ; 2.3.4.5.6. each for one single colony (486bp) ; 7. Postive control 

 

 

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