Difference between revisions of "Team:Tokyo Tech/Basic Part"

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      <h1>Basic Parts</h1>
 
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    <h4 class="subtitle"><strong>???</strong></h4>
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<h2 class="smalltitle">Tokyo Tech 2015 iGEM Team Basic Parts</h2>
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<p class="text"></p>
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<table width="900px" border="2" frame="border" style="border-collapse: collapse;"><tbody><tr height="20px" align="center"><th width="20%">Name</th><th width="10%">Type</th><th width="30%">Description</th><th width="20%">Design</th><th width="10%">Length(bp)</th><th width="10%">Experiment</th></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632000" target="_brank">BBa_K1632000</a></td><td width="10%">Regulatory</td><td width="30%"><i>fim</i> switch[default ON](Tokyo_Tech/J23119)</td><td width="20%">Riku Shinohara</td><td width="10%">382</td><td width="10%">Work</td></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632001" target="_brank">BBa_K1632001</a></td><td width="10%">Regulatory</td><td width="30%"><i>fim</i> switch[default ON](Tokyo_Tech/J23119)</td><td width="20%">Riku Shinohara</td><td width="10%">382</td><td width="10%">Work</td></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632004" target="_brank">BBa_K1632004</a></td><td width="10%">Regulatory</td><td width="30%"><i>fim</i> switch[default OFF](wild-type)</td><td width="20%">Riku Shinohara</td><td width="10%">382</td><td width="10%">Work</td></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632005" target="_brank">BBa_K1632005</a></td><td width="10%">Regulatory</td><td width="30%"><i>fim</i> switch[default OFF](wild-type)</td><td width="20%">Riku Shinohara</td><td width="10%">382</td><td width="10%">Work</td></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632006" target="_brank">BBa_K1632006</a></td><td width="10%">Regulatory</td><td width="30%"><i>fim</i> switch[default ON](Tokyo_Tech/B0010)</td><td width="20%">Riku Shinohara</td><td width="10%">597</td><td width="10%">Work</td></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632010" target="_brank">BBa_K1632010</a></td><td width="10%">Coding</td><td width="30%">fimB(wild-type)</td><td width="20%">Riku Shinohara</td><td width="10%">603</td><td width="10%">Work</td></tr>
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<tr height="20px"><td width="20%"><a href="http://parts.igem.org/Part:BBa_K1632011" target="_brank">BBa_K1632011</a></td><td width="10%">Coding</td><td width="30%">fimE(wild-type)</td><td width="20%">Riku Shinohara</td><td width="10%">597</td><td width="10%">Work</td></tr>
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</tbody></table></div>
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<p></p>
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<h2 class="smalltitle">1. <i>fim</i> switch (Tokyo_Tech): BBa_K1632000, BBa_K1632001, BBa_K1632007, BBa_K1632018, BBa_K1632019</h2>
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<ul>
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<li><a href="http://parts.igem.org/Part:BBa_K1632000" target="_brank">BBa_K1632000</a><br><i>fim</i> switch[default ON](Tokyo_Tech/J23119)</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1632001" target="_brank">BBa_K1632001</a><br><i>fim</i> switch[default OFF](Tokyo_Tech/J23119)</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1632006" target="_brank">BBa_K1632006</a><br><i>fim</i> switch[default ON](Tokyo_Tech/B0010)</li>
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</ul>
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</div>
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<p></p>
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<p class="text">We designed another <i>fim</i> switch with a standardized interchangeable promotor, <i>fim</i> switch (Tokyo_Tech).  A difference between the wild type <i>fim</i> switch and the <i>fim</i> switch (Tokyo_Tech) is that we replaced the sigma 70 promoter to the J23119 promotor" (<a href="http://parts.igem.org/Part:BBa_J23119" target="_brank">BBa_J23119</a>).  We also inserted two restriction enzyme sites in both the front (SalI and BamHI) and the back (BglII and MluI) of the promotor.  By inserting the restriction enzymes, our <i>fim</i> switch (Tokyo_Tech) turned into a <i>fim</i> switch with a standardized interchangeable promotor (Fig.5-1-1-1).  There is an example.  <a href="http://parts.igem.org/Part:BBa_K1632006" target="_brank">BBa_K1632006</a> is made by removing the J23119 promotor (<a href="http://parts.igem.org/Part:BBa_J23119" target="_brank">BBa_J23119</a>) and inserted Plac promotor (<a href="http://parts.igem.org/Part:BBa_B0010" target="_brank">BBa_B0010</a>) . </p>
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<p></p>
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<table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/c/cb/Tokyo_Tech_parts1.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-3-1-1. Design of Fim Switch (Tokyo_Tech)</h4></tr></td></tbody></table>
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<p><br><br></p>
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<div class="text">
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<ul>
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</ul>
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<p></p>
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<p></p>
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<h2 class="smalltitle">2. <i>fim</i> switch (wild-type): BBa_K1632004, BBa_K1632005 </h2>
 +
<p></p>
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<div class="text">
 +
<ul>
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<li><a href="http://parts.igem.org/Part:BBa_K1632004" target="_brank">BBa_K1632004</a><br><i>fim</i> switch[default ON](wild-type)</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1632005" target="_brank">BBa_K1632005</a><br><i>fim</i> switch[default OFF](wild-type)</li>
 +
</ul>
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</div>
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<p></p>
 +
<p></p>
 +
<p class="text">We are the first team in iGEM to successfully construct both the <i>fim</i> switch default state ON and the <i>fim</i> switch default state OFF and assay them.  These <i>fim</i> switch is derived from a wild type and the gene sequence is the same as that of a wild type E.coli.  The <i>fim</i> switch is inverted by the Fim recombinase.  Therefore, we can regulate the expression of the gene downstream of the <i>fim</i> switch by adding the Fim recombinase.  From the flow cytometers assay, they work ideally.</p>
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<p></p>
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<p><br><br></p>
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<p></p>
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<div class="text">
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<ul>
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</p>
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<p></p>
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<table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/e/e9/Tokyo_Tech_parts2.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-1-2-1. The result of our assay used <a href="http://parts.igem.org/Part:BBa_K1632007" target="_brank">BBa_K1632007</a>,<a href="http://parts.igem.org/Part:BBa_K1632008" target="_brank">BBa_K1632008</a> and <a href="http://parts.igem.org/Part:BBa_K1632013" target="_brank">BBa_K1632013</a> with flow cytometers</h4></td></tr></tbody></table>
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<p></p>
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<p></p>
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<p></p>
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<h2 class="smalltitle">3. Best New Basic Part: BBa_K1632012, BBa_K1632010</h2>
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<h3 class="sub5"> <a href="http://parts.igem.org/Part:BBa_K1632012" target="_brank">BBa_K1632012</a> meet the criteria of the Silver Medal</h3>
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<p></p>
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<div class="text">
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<ul>
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<li><a href="http://parts.igem.org/Part:BBa_K1632012" target="_brank">BBa_K1632012</a><br>Pbad/<i>araC</i>_fimB(wild-type)</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1632010" target="_brank">BBa_K1632010</a><br>fimB(wild-type)</li>
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</ul>
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</div>
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<p></p>
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<p></p>
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<p class="text">FimB (<a href="http://parts.igem.org/Part:BBa_K1632010" target="_brank">BBa_K1632010</a>) is a Fim recombinase.  This is derived from the wild type MG1655.  FimB invert the <i>fim</i> switch from the ON state to the OFF state and from the OFF state to the ON state (Fig.5-1-3-1.).</p>
 +
<p class="text">From our experimental results, we confirmed that the FimB protein inverts the <i>fim</i> switch in the ON-to-OFF direction and in the OFF-to-ON direction with approximately equal probability and works ideally (Fig.5-1-3-2.).  The expression of FimB is controlled by arabinose in <a href="http://parts.igem.org/Part:BBa_K1632012" target="_brank">BBa_K1632012</a>.</p>
 +
<table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/3/3f/Tokyo_Tech_parts6.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-1-3-1. <i>fim</i> switch is inverted by two recombinases, FimB and FimE. These proteins have distinct activities. The FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction with approximately equal probability</h4></td></tr></tbody></table>
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<p></p>
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<table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/9/9d/Tokyo_Tech_parts7.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-1-3-2. The result of our assay used <a href="http://parts.igem.org/Part:BBa_K1632007" target="_brank">BBa_K1632007</a>, <a href="http://parts.igem.org/Part:BBa_K1632008" target="_brank">BBa_K1632008</a> and <a href="http://parts.igem.org/Part:BBa_K1632012" target="_brank">BBa_K1632012</a> with flow cytometers.</h4></td></tr></tbody></table>
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<p></p>
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<h2 class="smalltitle">4. <i>fim</i>E (wild-type): BBa_K1632011 </h2>
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<p></p>
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<div class="text">
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 +
<li><a href="http://parts.igem.org/Part:BBa_K1632011" target="_brank">BBa_K1632011</a><br>FimE(wild-type)</li>
 +
</ul>
 +
</div>
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<p></p>
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<p></p>
 +
<p class="text">FimE(wild-type)(<a href="http://parts.igem.org/Part:BBa_K1632011" target="_brank">BBa_K1632011</a>) is Fim recombinases.  This Fim recombinase is derived from the wild type MG1655.  FimE invert the <i>fim</i> switch (wild-type) from the ON state to the OFF state.  The expression of this Fim recombinase is controlled by arabinose in <a href="http://parts.igem.org/Part:BBa_K1632013" target="_brank">BBa_K1632013</a>.  From our experimental results (Fig. 5-1-2-1.), they work ideally.
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Revision as of 13:25, 17 September 2015

Basic Parts

???

　　

Tokyo Tech 2015 iGEM Team Basic Parts

NameTypeDescriptionDesignLength(bp)Experiment
BBa_K1632000Regulatoryfim switch[default ON](Tokyo_Tech/J23119)Riku Shinohara382Work
BBa_K1632001Regulatoryfim switch[default ON](Tokyo_Tech/J23119)Riku Shinohara382Work
BBa_K1632004Regulatoryfim switch[default OFF](wild-type)Riku Shinohara382Work
BBa_K1632005Regulatoryfim switch[default OFF](wild-type)Riku Shinohara382Work
BBa_K1632006Regulatoryfim switch[default ON](Tokyo_Tech/B0010)Riku Shinohara597Work
BBa_K1632010CodingfimB(wild-type)Riku Shinohara603Work
BBa_K1632011CodingfimE(wild-type)Riku Shinohara597Work

1. fim switch (Tokyo_Tech): BBa_K1632000, BBa_K1632001, BBa_K1632007, BBa_K1632018, BBa_K1632019

We designed another fim switch with a standardized interchangeable promotor, fim switch (Tokyo_Tech). A difference between the wild type fim switch and the fim switch (Tokyo_Tech) is that we replaced the sigma 70 promoter to the J23119 promotor" (BBa_J23119). We also inserted two restriction enzyme sites in both the front (SalI and BamHI) and the back (BglII and MluI) of the promotor. By inserting the restriction enzymes, our fim switch (Tokyo_Tech) turned into a fim switch with a standardized interchangeable promotor (Fig.5-1-1-1). There is an example. BBa_K1632006 is made by removing the J23119 promotor (BBa_J23119) and inserted Plac promotor (BBa_B0010) .

Fig.5-3-1-1. Design of Fim Switch (Tokyo_Tech)



2. fim switch (wild-type): BBa_K1632004, BBa_K1632005

We are the first team in iGEM to successfully construct both the fim switch default state ON and the fim switch default state OFF and assay them. These fim switch is derived from a wild type and the gene sequence is the same as that of a wild type E.coli. The fim switch is inverted by the Fim recombinase. Therefore, we can regulate the expression of the gene downstream of the fim switch by adding the Fim recombinase. From the flow cytometers assay, they work ideally.



    Fig.5-1-2-1. The result of our assay used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers

    3. Best New Basic Part: BBa_K1632012, BBa_K1632010

    BBa_K1632012 meet the criteria of the Silver Medal

    FimB (BBa_K1632010) is a Fim recombinase. This is derived from the wild type MG1655. FimB invert the fim switch from the ON state to the OFF state and from the OFF state to the ON state (Fig.5-1-3-1.).

    From our experimental results, we confirmed that the FimB protein inverts the fim switch in the ON-to-OFF direction and in the OFF-to-ON direction with approximately equal probability and works ideally (Fig.5-1-3-2.). The expression of FimB is controlled by arabinose in BBa_K1632012.

    Fig.5-1-3-1. fim switch is inverted by two recombinases, FimB and FimE. These proteins have distinct activities. The FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction with approximately equal probability

    Fig.5-1-3-2. The result of our assay used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers.

    4. fimE (wild-type): BBa_K1632011

  • BBa_K1632011
    FimE(wild-type)

FimE(wild-type)(BBa_K1632011) is Fim recombinases. This Fim recombinase is derived from the wild type MG1655. FimE invert the fim switch (wild-type) from the ON state to the OFF state. The expression of this Fim recombinase is controlled by arabinose in BBa_K1632013. From our experimental results (Fig. 5-1-2-1.), they work ideally.