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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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- 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.
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- 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.
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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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