Team:UC Davis/notebook/c0051debug.html

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Revision as of 10:11, 27 October 2010

Characterization of BBa_C0051, one of the most widely used and studied parts

Bba_C0051 cI-Lamba, the promoter in disguise During the construction of one of our larger pieces of the spatial oscillator, the ON switch, we stumbled upon an unexpected phenotype.

The Problem
  • Our intermediate ligation of RBS-C0051 to RBS-RFP produced a red phenotype but it has no promoter!
  • Since Bba_C0051 is a highly studied and widely used part in the registry, it presents a huge problem not only in the context of the spatial oscillator but also in the 76 parts that utilize it throughout the registry.
  • This exciting new find causes many problems in our design and must cause malfunctions in a huge number of parts entered and used in the registry.


The Investigation

  • We began by sequencing the DNA of the red colonies from the ligation of RBS-C0051 to RBS-RFP in order to rule out the presence of any promoter or mutation. The sequence analysis revealed that we did indeed have the correct sequence.
  • Further investigation into Bba_C0051 showed that the part not only contained the coding region, cI, but it is also followed by an LVA degradation tag and a barcode. When looking further into the RBS (Bba_E1010), we found that it also contained an LVA degradation tag and a barcode after the RFP coding region.
  • We then conducted a number of experiments in order to determine which part or combination of parts contained in the construct caused this unwanted promoter-like behavior.
  • After determining the cause of the unwanted transcription, we continued to conduct a number of experiments in order to measure the results.


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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