Difference between revisions of "Team:Jiangnan China/Parts"

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{{Jiangnan_China}}
 
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        <a class="nav-link py-2 d-none d-md-inline-block" href="https://2018.igem.org/Team:Jiangnan_China/InterLab"><i class="fa fa-interlab" aria-hidden="true"></i> InterLab</a>
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<h1>Parts</h1>
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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<h3>Note</h3>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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      <img src="https://static.igem.org/mediawiki/2018/5/57/T--jiangnan_china--part--a.jpg" width="100%">
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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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<h3>Adding parts to the registry</h3>
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      <div class="dcpt3" style="font-size:20px;line-height:1.5;font-family: 'spr';margin-top: -1px">
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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    <div align="left" style="font-family: 'spr';font-size:50px;border-bottom:2px solid #584b4f;"><strong>Basic part</strong></div>
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      <br>
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      <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>
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      <br>
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      &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>
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      &nbsp;&nbsp;&nbsp;&nbsp;<strong><font size="5">Usage and function</font></strong><br>
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    &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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    <div align="center">
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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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    </div>
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<div align="center">
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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>
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    <div style="text-align:center;"><strong >Fig 1</strong> Survival rate at acid stress (pH 4.0).</div><br>
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    &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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    <div align="center">
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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>
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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>
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    &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>
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    <br>
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      <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>
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      <br>
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      &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>
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      &nbsp;&nbsp;&nbsp;&nbsp;Kegg number: LLNZ_06470.<br>
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      <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>
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      <div align="center">
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      <img src="https://static.igem.org/mediawiki/2018/b/b7/T--jiangnan_china--wet--11.png" width="60%" >
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    </div>
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    <div style="text-align:center;"><strong >Fig 3</strong> Electron microscopy before and after repeated freezing and thawing.<br><br></div>
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    &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>
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    <br>
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      <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>
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      <br>
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      &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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      <div align="center">
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      <img src="https://static.igem.org/mediawiki/2018/c/c4/T--jiangnan_china--part--5.png" width="30%" >
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    </div>
  
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
 
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<a href="http://parts.igem.org/Add_a_Part_to_the_Registry">
 
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ADD PARTS
 
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<h3>Inspiration</h3>
 
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
 
  
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
 
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
 
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
 
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      <a name="Composite"></a>
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        <br>
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    <div align="left" style="font-family: 'spr';font-size:50px;border-bottom:2px solid #584b4f;"><strong>Composite part</strong></div>
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    <br>
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      <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>
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      <br>
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      &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.
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      <br><br>
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      &nbsp;&nbsp;&nbsp;&nbsp;<strong><font size="5">Usage and biology</font></strong><br>
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      &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>
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      &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.
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      <br><br>
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      &nbsp;&nbsp;&nbsp;&nbsp;<strong><font size="5">Acid stress </font></strong><br>
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        &nbsp;&nbsp;&nbsp;&nbsp;Deal with the samples under pH 4.0 with nisin as an inducer.<br>
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        <div align="center">
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      <img src="https://static.igem.org/mediawiki/2018/6/62/T--jiangnan_china--wet--7.png" width="80%" >
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    </div>
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    <div style="text-align:center;"><strong >Fig 4</strong> Number of colonies at acid stress (pH 4.0).<br><br></div>
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    <div align="center">
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      <img src="https://static.igem.org/mediawiki/2018/7/71/T--jiangnan_china--wet--8.png" width="80%" >
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    </div>
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<div align="center">
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      <img src="https://static.igem.org/mediawiki/2018/6/66/T--jiangnan_china--yl--2.png" width="50%" >
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    </div>
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    <div style="text-align:center;"><strong >Fig 5</strong> Survival rate at acid stress (pH4.0).<br><br></div>
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    &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>
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    <div align="center">
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      <img src="https://static.igem.org/mediawiki/2018/f/fb/T--jiangnan_china--wet--10.jpg" width="50%" >
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    </div>
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    <div style="text-align:center;"><strong >Fig 8</strong> The Comparison curve of survival rate under cold stress.<br><br></div>
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    &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>
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<h3>What information do I need to start putting my parts on the Registry?</h3>
 
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<p>The information needed to initially create a part on the Registry is:</p>
 
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<ul>
 
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<li>Part Name</li>
 
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<li>Part type</li>
 
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<li>Creator</li>
 
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<li>Sequence</li>
 
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<li>Short Description (60 characters on what the DNA does)</li>
 
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<li>Long Description (Longer description of what the DNA does)</li>
 
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<li>Design considerations</li>
 
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<p>
 
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
 
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<h3>Part Table </h3>
 
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<p>Please include a table of all the parts your team has made during your project on this page. Remember part characterization and measurement data must go on your team part pages on the Registry. </p>
 
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Latest revision as of 12:41, 17 October 2018


Basic part

● msmK (BBa_K2606001)
    It is a fragment cloned from Lactococcus lactis NZ9000 gene group, which is verified to be a new and effective anti-acid gene in Lactococcus lactis NZ3900.

    Usage and function
    The msmK overexpression strain has 213-fold higher survival rate than parent one at pH 4.0 after 3 hours.

Fig 1 Survival rate at acid stress (pH 4.0).

    The result shows that msmK is an effective anti-acid gene, which can make the recombinant strain has 213-fold higher survival rate than parent one.

Fig 2 Electron microscopy of L.lactis NZ3900/pNZ8149-msmk-cspD2-gfp and L.lactis NZ3900/pNZ8149 before and after acid stress.

    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.

● cspD2 (BBa_K2606003)
    The cspD2 gene is a reported that can express cold shock protein in Lactococcus lactis, and can help bacteria survive at low temperature.
    Kegg number: LLNZ_06470.

Fig 3 Electron microscopy before and after repeated freezing and thawing.

    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.

● gfp (BBa_K2606004)
    It can express green fluorescent protein. We use it as a marker gene in the process to characterize the number of cells.

Composite part

● msmK-cspD2 (BBa_K2606005)
    It is the final composite biobrick of our project, we overexpress anti-acid gene msmK and anti-cold gene cspD2 via the NICE system.

    Usage and biology
    The gene cspD2 was ligated to the Pnz8149/msmK plasmid by one-step cloning (seamless ligation), and the recombinant plasmid was introduced into the constructed L. lactis NZ3900/pNZ8149-msmk-cspD2 strain using electroporation.
    The gfp 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.

    Acid stress
    Deal with the samples under pH 4.0 with nisin as an inducer.
Fig 4 Number of colonies at acid stress (pH 4.0).

Fig 5 Survival rate at acid stress (pH4.0).

    The result shows that composite part can make the recombinant strain has 243-fold higher survival rate than parent one under acid stress.
Fig 8 The Comparison curve of survival rate under cold stress.

    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 msmK-cspD2.

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