Team:UC Davis/notebook/c0051debug.html

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<a name="test1"></a><p class="header"><b>Test #1</b></a><p>
<a name="test1"></a><p class="header"><b>Test #1</b></a><p>
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     <li>To determine whether Bba_C0051 played a role in the unexpected transcription, we ligated RBS-RFP to RBS-RFP in order to see if transcription occurred. </li>
     <li>To determine whether Bba_C0051 played a role in the unexpected transcription, we ligated RBS-RFP to RBS-RFP in order to see if transcription occurred. </li>
     <li>The construct exhibited a white phenotype. Thus, we concluded that transcription did not start in any part of the RFP. This helped to narrow our focus down to the possibility of the cI coding region in Bba_C0051 or a possible artifact in the plasmid as the cause for the unwanted transcription.</li>
     <li>The construct exhibited a white phenotype. Thus, we concluded that transcription did not start in any part of the RFP. This helped to narrow our focus down to the possibility of the cI coding region in Bba_C0051 or a possible artifact in the plasmid as the cause for the unwanted transcription.</li>
     <li>After the results were in, we diligently sent them out for sequencing so that we would be sure that our results were accurately reflecting the parts that we believed we were analyzing.</li>
     <li>After the results were in, we diligently sent them out for sequencing so that we would be sure that our results were accurately reflecting the parts that we believed we were analyzing.</li>
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<a name="test2"></a><p class="header"><b>Test #2</b></a><p>
<a name="test2"></a><p class="header"><b>Test #2</b></a><p>

Revision as of 11:00, 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 scanned image of our plate with colonies containing RBS-C0051 ligated to RBS-RFP. Click above for the full-screen image.
  • 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.


A Starting Point

Our scanned image of our plate with colonies containing RBS-RFP. No abnormalities here! Click to enlarge.
  • The previous step in the construction of the on switch was RBS-RFP.
  • This construct clearly exhibited a white phenotype. No abnormalities here!


Test #1
  • To determine whether Bba_C0051 played a role in the unexpected transcription, we ligated RBS-RFP to RBS-RFP in order to see if transcription occurred.
  • The construct exhibited a white phenotype. Thus, we concluded that transcription did not start in any part of the RFP. This helped to narrow our focus down to the possibility of the cI coding region in Bba_C0051 or a possible artifact in the plasmid as the cause for the unwanted transcription.
  • After the results were in, we diligently sent them out for sequencing so that we would be sure that our results were accurately reflecting the parts that we believed we were analyzing.


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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