Team:UC Davis/notebook/c0051debug.html

From 2010.igem.org

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<li><a href="#test3" class="help">Test #3</a></li>
<li><a href="#test3" class="help">Test #3</a></li>
<li><a href="5race" class="help">5' Race PCR</a></li>
<li><a href="5race" class="help">5' Race PCR</a></li>
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<li><a href="#tets4" class="help">Tets #4</a></li>
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<li><a href="#tets4" class="help">Test #4</a></li>
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Revision as of 21:27, 24 October 2010

BBa_C0051 Debug

The Problem

  • When we ligated RBS-C0051 to RBS-RFP together, we noticed that there were RED colonies.
  • There is no promoter present yet. How this happened was a mystery that we had to look into.


The Investigation

  • First of all, we sequenced the DNA of the red colonies. The sequence analysis showed that we indeed had the correct sequence.
  • We decided to run a number of tests to see which part of the sequence exhibited promoter-like behavior.
  • Unwanted transcription may be due to the nature of the construct.
  • Promoter-like sequence may be present anywhere in the BBa_C0051 or BBa_E1010 (RFP) biobrick, including the LVA and bar code.


Test #1

  • We ligated RBS-RFP to RBS-RFP to see if transcription occurred.
  • The construct exhibited a white phenotype. Thus, we concluded that transcription didn't start in any part of the RFP

Test #2

  • We ligated RBS-RFP to Chris Anderson's Promoter Screening Plasmid.
  • We also obtained white colonies.


Test #3

  • We ligated RBS-C0051 to Chris Anderson's Promoter Screening Plasmid.
  • And we have obtained red colonies! We know that the transcription initiation is somewhere in the BBa_C0051 region.

5' Race PCR

  • To determine where transcription is initiating, we have decided to perform 5' Race PCR on select regions of c0051


Test #4

  • Surely enough, after ligating the C0051 without the barcode into the Chris Anderson promoter screening plasmid, we have obtained white colonies.


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! :)

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