Team:UC Davis/notebook/c0051debug.html

From 2010.igem.org

(Difference between revisions)

Revision as of 21:25, 24 October 2010

	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.

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.

	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

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

	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.

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

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


The Problem The Investigation Test #1 Test #2 Test #3 5' Race PCR Tets #4

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! :)

@@ Line 169: / Line 169: @@
 <li><a href="#c0051debug" class="help">The Problem</a></li>
 <li><a href="#investigation" class="help">The Investigation</a></li>
-<li><a href="#test1" class="help">Test 1</a></li>
+<li><a href="#test1" class="help">Test #1</a></li>
-<li><a href="#test2" class="help">Test 2</a></li>
+<li><a href="#test2" class="help">Test #2</a></li>
-<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="#tets4" class="help">Tets 4</a></li>
+<li><a href="#tets4" class="help">Tets #4</a></li>
 </ul> </td>
                 </tr>