Team:UC Davis/notebook/assembly.html

From 2010.igem.org

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<a name="assembly"></a><p class="header"><b>Assembly Workplans</b></a><p>
<a name="assembly"></a><p class="header"><b>Assembly Workplans</b></a><p>
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<a name="misc"></a><p class="header"><b>Miscellaneous Parts</b></a><p>
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<a name="pieces"></a><p class="header"><b>Creating Each Individual Composite Part</b></a><p>
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<b>**Click on the individual pieces below to view how we ligated them together.**</b> <p><br />
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<b>Miscellaneous Parts</b></a><br />
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<a name="circuit"></a><p class="header"><b>Genetic Circuit</b></a><p>
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<b>Genetic Circuit Pieces</b><br />
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<b>**Click on the individual pieces above to view how we ligated them together.**</b> <p><br />
 
<a name="plasmid"></a><p class="header"><b>Next Mission: Placing them into only two plasmids</b></a><p>
<a name="plasmid"></a><p class="header"><b>Next Mission: Placing them into only two plasmids</b></a><p>
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<p class="header"><b>Sample Ligation Procedure</b></a><p>  
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<a name="topagar"></a><p class="header"><b>Final Mission: Create an Even Lawn of E. Coli</b><br />
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<img src="https://static.igem.org/mediawiki/2010/2/29/Initiatorconstruct.jpg"><p>
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<ul>
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<li>We are using this <a href="https://2010.igem.org/Team:UC_Davis/protocols/topagar.html" class="help">top agar plating technique </a></li>
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<li>Next is to test the what the optimal OD, arabinose concentration, and amount of light it takes to a) create an even lawn of E. Coli and b) see contrast between the "on" and "off" cells </li>
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</ul><p><br />
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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>
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<a name="protocols"></a><p class="header"><b>Basic Assembly Protocols</b></a><p>
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<b>For more insight about the process behind the ligations, here is a sample ligation procedure.  Because the initiator is one of the smaller constructs, we have decided to use it as an example to illustrate the protocols used in lab.</b><p>
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<img src="https://static.igem.org/mediawiki/2010/2/29/Initiatorconstruct.jpg"><p>
<a name="startup"><h1>Starting Up</h1></a>
<a name="startup"><h1>Starting Up</h1></a>
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<li><a href="#misc" class="help">Miscellaneous Parts</li>
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<li><a href="#assembly" class="help">Assembly Workplan</li>
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<li><a href="#circuit" class="help">Main Genetic Circuit</li>
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<li><a href="#pieces" class="help">Creating Each Composite Part</li>
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<li><a href="#sample" class="help">Sample Ligation Procedure</li>
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<li><a href="#plasmid" class="help">Placing Into Two Plasmids</li>
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<li><a href="#topagar" class="help">Creating Lawn of E. Coli</li>
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<li><a href="#protocols" class="help">Basic Assembly Protocols</li>
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Latest revision as of 22:19, 27 October 2010

Assembly Workplans

Creating Each Individual Composite Part

**Click on the individual pieces below to view how we ligated them together.**


Miscellaneous Parts


Genetic Circuit Pieces


Next Mission: Placing them into only two plasmids



Final Mission: Create an Even Lawn of E. Coli

  • We are using this top agar plating technique
  • Next is to test the what the optimal OD, arabinose concentration, and amount of light it takes to a) create an even lawn of E. Coli and b) see contrast between the "on" and "off" cells


Basic Assembly Protocols

For more insight about the process behind the ligations, here is a sample ligation procedure. Because the initiator is one of the smaller constructs, we have decided to use it as an example to illustrate the protocols used in lab.

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! :)