From 2010.igem.org
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| - | <li>When we ligated RBS-C0051 to RBS-RFP together, we noticed that there were RED colonies.</li> | + | <li>Our intermediate ligation of RBS-C0051 to RBS-RFP produced a red phenotype but it has no promoter! </li> |
| - | <li>There is no promoter present yet. How this happened was a mystery that we had to look into.</li> | + | <li>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.</li> |
| | + | <li>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. </li> |
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| - | <li>First of all, we sequenced the DNA of the red colonies. The sequence analysis showed that we indeed had the correct sequence.</li> | + | <li>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.</li> |
| - | <li>We decided to run a number of tests to see which part of the sequence exhibited promoter-like behavior.</li> | + | <li>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. </li> |
| - | <li>Unwanted transcription may be due to the nature of the construct.</li> | + | <li>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.</li> |
| - | <li>Promoter-like sequence may be present anywhere in the BBa_C0051 or BBa_E1010 (RFP) biobrick, including the LVA and bar code. </li> | + | <li>After determining the cause of the unwanted transcription, we continued to conduct a number of experiments in order to measure the results.</li> |
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Revision as of 09:54, 27 October 2010
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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.
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- 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.
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- 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.
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- 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
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- We ligated RBS-RFP to Chris Anderson's Promoter Screening Plasmid.
- We also obtained white colonies.
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- 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.
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- To determine where transcription is initiating, we have decided to perform 5' Race PCR on select regions of c0051
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- Surely enough, after ligating the C0051 without the barcode into the Chris Anderson promoter screening plasmid, we have obtained white colonies.
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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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