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      <div id="dcpt4" style="font-size:20px;line-height:1.5;font-family: 'spr';">
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      <img src="https://static.igem.org/mediawiki/2018/5/5a/T--jiangnan_china--part--1.png" width="70%" >
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     <div align="left" style="font-family: 'spr';font-size:50px;border-bottom:2px solid #584b4f;"><strong>Basic part</strong></div>
      <br>
      <strong ><font size="6">● <span class="font-italic">msmK</span> (<a href="http://parts.igem.org/Part:BBa_K2606001" target="_blank">BBa_K2606001</a>)</font></strong>
      <br>
      &nbsp;&nbsp;&nbsp;&nbsp;It is a fragment cloned from <span class="font-italic">Lactococcus lactis</span> NZ9000 gene group, which is verified to be a new and effective anti-acid gene in <span class="font-italic">Lactococcus lactis</span> NZ3900.
      <br><br>
      &nbsp;&nbsp;&nbsp;&nbsp;<strong><font size="5">Usage and function</font></strong><br>
    &nbsp;&nbsp;&nbsp;&nbsp;The <span class="font-italic">msmK</span> overexpression strain has 213-fold higher survival rate than parent one at pH 4.0 after 3 hours.<br><br>
    
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      <img src="https://static.igem.org/mediawiki/2018/0/0f/T--jiangnan_china--wet--6.png" width="60%" >
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      <img src="https://static.igem.org/mediawiki/2018/8/88/T--jiangnan_china--yl--1.png" width="50%" >
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     <div style="text-align:center;"><strong >Fig 1</strong> Survival rate at acid stress (pH 4.0).</div><br>
     &nbsp;&nbsp;&nbsp;&nbsp;The result shows that <span class="font-italic">msmK</span> is an effective anti-acid gene, which can make the recombinant strain has 213-fold higher survival rate than parent one.<br><br>
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      <img src="https://static.igem.org/mediawiki/2018/9/9f/T--jiangnan_china--wet--9.png" width="60%" >
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     <div style="text-align:center;"><strong >Fig 2</strong> Electron microscopy of <span class="font-italic">L.lactis</span> NZ3900/pNZ8149-<span class="font-italic">msmk-cspD2-gfp</span> and <span class="font-italic">L.lactis</span> NZ3900/pNZ8149 before and after acid stress.<br><br></div>
     &nbsp;&nbsp;&nbsp;&nbsp;Before the acid stress, the cell structure of the control strain and the recombinant strain remained intact. After 3 hours of pH 4.0 stress, the cell membrane thickness became thinner and the surface became rough, and the cell membrane of some control strains ruptured. In comparison, the cell structure of the recombinant strain remains more intact, thereby effectively reducing the damage caused by acid stress on the cells.<br>
     <br>
      <strong ><font size="6">● <span class="font-italic">cspD2</span> (<a href="http://parts.igem.org/Part:BBa_K2606003" target="_blank">BBa_K2606003</a>)</font></strong>
      <br>
      &nbsp;&nbsp;&nbsp;&nbsp;The <span class="font-italic">cspD2</span> gene is a reported that can express cold shock protein in <span class="font-italic">Lactococcus lactis</span>, and can help bacteria survive at low temperature.<br>
      &nbsp;&nbsp;&nbsp;&nbsp;Kegg number: LLNZ_06470.<br>
      <div style="text-align:center"><a href="https://www.genome.jp/dbget-bin/www_bget?lln:LLNZ_06470" target="_blank">https://www.genome.jp/dbget-bin/www_bget?lln:LLNZ_06470</a></div><br>
      <div align="center">
      <img src="https://static.igem.org/mediawiki/2018/b/b7/T--jiangnan_china--wet--11.png" width="60%" >
     </div>
     <div style="text-align:center;"><strong >Fig 3</strong> Electron microscopy before and after repeated freezing and thawing.<br><br></div>
     &nbsp;&nbsp;&nbsp;&nbsp;Before freezing and thawing, the cell structure of the control strain and the recombinant strain remained intact. After repeated freezing and thawing for 4 times, the cell membrane of the cell became thin and rough, and some intracellular substances overflowed. In comparison, the cell structure of the recombinant strain remains more intact, and the damage of the cell membrane is alleviated, thereby effectively reducing the damage caused by freezing stress on the cells.<br>
     <br>
      <strong ><font size="6">● <span class="font-italic">gfp</span> (<a href="http://parts.igem.org/Part:BBa_K2606004" target="_blank">BBa_K2606004</a>)</font></strong>
      <br>
      &nbsp;&nbsp;&nbsp;&nbsp;It can express green fluorescent protein. We use it as a marker gene in the process to characterize the number of cells.<br>
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      <img src="https://static.igem.org/mediawiki/2018/c/c4/T--jiangnan_china--part--5.png" width="30%" >
     </div>









      </div>
      <a name="Composite"></a>
      <div id="dcpt4" style="font-size:20px;line-height:1.5;font-family: 'spr';">
        <br>
     <div align="left" style="font-family: 'spr';font-size:50px;border-bottom:2px solid #584b4f;"><strong>Composite part</strong></div>
     <br>
      <strong ><font size="6">● <span class="font-italic">msmK-cspD2</span> (<a href="http://parts.igem.org/Part:BBa_K2606005" target="_blank">BBa_K2606005</a>)</font></strong>
      <br>
      &nbsp;&nbsp;&nbsp;&nbsp;It is the final composite biobrick of our project, we overexpress anti-acid gene <span class="font-italic">msmK</span> and anti-cold gene <span class="font-italic">cspD2</span> via the NICE system.
      <br><br>
      &nbsp;&nbsp;&nbsp;&nbsp;<strong><font size="5">Usage and biology</font></strong><br>
      &nbsp;&nbsp;&nbsp;&nbsp;The gene <span class="font-italic">cspD2</span> was ligated to the Pnz8149/<span class="font-italic">msmK</span> plasmid by one-step cloning (seamless ligation), and the recombinant plasmid was introduced into the constructed L. lactis NZ3900/pNZ8149-<span class="font-italic">msmk-cspD2</span> strain using electroporation. <br>
      &nbsp;&nbsp;&nbsp;&nbsp;The <span class="font-italic">gfp</span> gene was inserted as a marker gene, and cell viability was characterized by fluorescence intensity. The strain was tested for acid resistance and freezing resistance using a flow cytometer. The process of acid stress and cold stress is similar with the above demonstration process.
      <br><br>
      &nbsp;&nbsp;&nbsp;&nbsp;<strong><font size="5">Acid stress </font></strong><br>
        &nbsp;&nbsp;&nbsp;&nbsp;Deal with the samples under pH 4.0 with nisin as an inducer.<br>
        <div align="center">
      <img src="https://static.igem.org/mediawiki/2018/6/62/T--jiangnan_china--wet--7.png" width="80%" >
     </div>
     <div style="text-align:center;"><strong >Fig 4</strong> Number of colonies at acid stress (pH 4.0).<br><br></div>
     <div align="center">
      <img src="https://static.igem.org/mediawiki/2018/7/71/T--jiangnan_china--wet--8.png" width="80%" >
     </div>
<div align="center">
      <img src="https://static.igem.org/mediawiki/2018/6/66/T--jiangnan_china--yl--2.png" width="50%" >
     </div>
     <div style="text-align:center;"><strong >Fig 5</strong> Survival rate at acid stress (pH4.0).<br><br></div>
     &nbsp;&nbsp;&nbsp;&nbsp;The result shows that composite part can make the recombinant strain has 243-fold higher survival rate than parent one under acid stress.<br>
     <div align="center">
      <img src="https://static.igem.org/mediawiki/2018/f/fb/T--jiangnan_china--wet--10.jpg" width="50%" >
     </div>
     <div style="text-align:center;"><strong >Fig 8</strong> The Comparison curve of survival rate under cold stress.<br><br></div>
     &nbsp;&nbsp;&nbsp;&nbsp;After 4 consecutive repeated freeze-thaw tests, the recombinant strain was 47.5 times more viable than the control strain, indicating the antifreeze survival rate of the strain with increased overexpression of <span class="font-italic">msmK-cspD2</span>.<br> 
      </div>

      

    



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