Team:UC Davis/notebook/initiator.html

From 2010.igem.org

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         <table class="pikachu" width="690px">
         <table class="pikachu" width="690px">
             <tr><td class="kirby">
             <tr><td class="kirby">
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<p class="header"><b>The Initiator</b></a><p>     
+
<p class="header"><b>Assembly Workplan: The Initiator</b></a><p>     
 +
 
 +
<img src="https://static.igem.org/mediawiki/2010/2/29/Initiatorconstruct.jpg"><p>
 +
 
 +
Because the initiator is one of the smaller constructs, we have decided to use this as an example to illustrate the utmost basic workplan in greater detail. <p>
<a name="startup"><h1>Starting Up</h1></a>
<a name="startup"><h1>Starting Up</h1></a>
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</ul><p><br />
</ul><p><br />
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<a name="rbsc0051"><h1>Ligating RBS to BBa_C0051</h1></a>
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<a name="rbsc0051"><h1>RBS to BBa_C0051</h1></a>
<ul>
<ul>
-
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells with BBa_B0034 and BBa_C0051</li>
+
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: BBa_B0034 and BBa_C0051</li>
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> BBa_B0034 and BBa_C0051</li>
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> BBa_B0034 and BBa_C0051</li>
</ul>
</ul>
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  </td>
  </td>
</tr>
</tr>
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</table><p>
+
</table><p><br />
 +
 
 +
<ul>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the RBS and BBa_C0051 fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on carb plates.</li>
 +
<li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed.
 +
</ul><br />
 +
 
 +
<img src="https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg"><br />
 +
 
 +
Assembly notes
 +
<ul>
 +
<li>It is most practical to cut RBS as a vector because RBS is less than 100 bp long as an insert, making it hard to see and extract on the gel.</li>
 +
<li>The sticky ends from the XbaI and SpeI, when ligated together, will form an 8 bp scar.  Thus, XbaI and SpeI will no longer be able to cut here.</li>
 +
</ul><br />
 +
 
 +
<a name="stop"><h1>'RBS & BBa_C0051' to Terminator</h1></a>
 +
<ul>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: 'RBS & BBa_C0051', BBa_B0015</li>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> 'RBS & BBa_C0051' and BBa_B0015</li>
 +
</ul>
 +
 
 +
<table border="0" margin="1" width="650px" padding="5px">
 +
<tr>
 +
<td>
 +
    <table border="0" margin="1" width="320px" padding="5px">
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/9/9a/Stopplasmid.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with EcoRI & XbaI</li></ul></td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/7/70/Xstopopenvector.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul>
 +
        <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li>
 +
        <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/7/70/Xstopopenvector.jpg"></td>
 +
    </tr>
 +
    </table>
 +
</td>
 +
<td>
 +
  <table border="0" margin="1" width="320px" padding="5px">
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with EcoRI & SpeI</li></ul> </td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/9/94/RBSc0051cut.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/d/de/RBSc0051insertx.jpg"></td>
 +
    </tr>
 +
  </table>
 +
</td>
 +
</tr>
 +
</table><p><br />
 +
 
 +
<ul>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on kan plates.</li>
 +
<li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed.
 +
</ul><br />
 +
 
 +
<img src="https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg"><br /><p>
 +
 
 +
Assembly notes <br />
 +
<ul>
 +
<li>It is most practical to cut BBa_B0015 as the vector because it is only 130 bp long.  It would be hard to see on a gel.  </li>
 +
</ul><br />
 +
 
 +
<a name="r0082"><h1>'RBS & BBa_C0051 & stop' to BBa_R0082</h1></a>
 +
<ul>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: 'RBS & BBa_C0051 & stop', BBa_R0082</li>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> 'RBS & BBa_C0051& stop' and BBa_R0082</li>
 +
</ul>
 +
 
 +
<table border="0" margin="1" width="650px" padding="5px">
 +
<tr>
 +
<td>
 +
    <table border="0" margin="1" width="320px" padding="5px">
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/c/cc/Promoterclosed.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with SpeI & PstI</li></ul></td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/2/21/Promoterplasmidopenx.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul>
 +
        <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li>
 +
        <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/2/21/Promoterplasmidopenx.jpg"></td>
 +
    </tr>
 +
    </table>
 +
</td>
 +
<td>
 +
  <table border="0" margin="1" width="320px" padding="5px">
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with XbaI & PstI</li></ul> </td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/4/46/RBSc0051stopcut.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td>
 +
    </tr>
 +
    <tr>
 +
      <td><img src="https://static.igem.org/mediawiki/2010/6/61/RBSc0051stopinsertx.jpg"></td>
 +
    </tr>
 +
  </table>
 +
</td>
 +
</tr>
 +
</table><p><br />
 +
 
 +
<ul>
 +
<li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on carb plates.</li>
 +
<li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed.
 +
</ul><br />
 +
 
 +
<img src="https://static.igem.org/mediawiki/2010/3/34/Initiatorplasmid.jpg"><br />
 +
 
 +
Assembly notes
 +
<ul>
 +
<li>The promoter had to be cut as a vector because it is only 108 bp by itself.</li>
 +
</ul><br />
                            
                            
         </td>
         </td>
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<ul>
<ul>
<li><a href="#startup" class="help">Starting Up</a></li>
<li><a href="#startup" class="help">Starting Up</a></li>
-
<li><a href="#rbsc0051" class="help">Ligating RBS to c0051</a></li>
+
<li><a href="#rbsc0051" class="help">RBS to c0051</a></li>
 +
<li><a href="#stop" class="help">'RBS & c0051' to stop</a></li>
 +
<li><a href="#r0082" class="help">'RBS & c0051 & stop' to R0082</a></li>
</ul> </td>
</ul> </td>
               </tr>
               </tr>

Latest revision as of 11:35, 19 September 2010

Assembly Workplan: The Initiator

Because the initiator is one of the smaller constructs, we have decided to use this as an example to illustrate the utmost basic workplan in greater detail.

Starting Up

  • Hydrated parts: BBa_B0034, BBa_C0051, BBa_B0015, BBa_R0082.
  • Transformed these parts into DH5α competent cells.


RBS to BBa_C0051


  • Ligate the RBS and BBa_C0051 fragments together and transform on carb plates.
  • Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.


Assembly notes
  • It is most practical to cut RBS as a vector because RBS is less than 100 bp long as an insert, making it hard to see and extract on the gel.
  • The sticky ends from the XbaI and SpeI, when ligated together, will form an 8 bp scar. Thus, XbaI and SpeI will no longer be able to cut here.

'RBS & BBa_C0051' to Terminator
  • Cultured cells that have parts: 'RBS & BBa_C0051', BBa_B0015
  • Miniprepped 'RBS & BBa_C0051' and BBa_B0015


  • Ligate the fragments together and transform on kan plates.
  • Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.


Assembly notes

  • It is most practical to cut BBa_B0015 as the vector because it is only 130 bp long. It would be hard to see on a gel.

'RBS & BBa_C0051 & stop' to BBa_R0082
  • Cultured cells that have parts: 'RBS & BBa_C0051 & stop', BBa_R0082
  • Miniprepped 'RBS & BBa_C0051& stop' and BBa_R0082


  • Ligate the fragments together and transform on carb plates.
  • Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.


Assembly notes
  • The promoter had to be cut as a vector because it is only 108 bp by itself.
We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

Retrieved from "http://2010.igem.org/Team:UC_Davis/notebook/initiator.html"