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Team:Illinois/Hybrid Promoter
From 2009.igem.org
Contents |
Hybrid Promoter
Goals: The goal of this side-project is the create a hybrid or combinatorial promoter that accepts two inputs. This will be used in the creation of an AND logic gate.
Papers of Interest:
[http://www.nature.com/nature/journal/v420/n6912/full/nature01257.html Engineered gene circuits] Jeff Hasty, David McMillen & J. J. Collins
[http://aem.asm.org/cgi/content/abstract/75/3/637 Construction and Enhancement of a Minimal Genetic AND Logic Gate] Daniel J. Sayut, Yan Niu, and Lianhong Sun
[http://www.sciencemag.org/cgi/content/full/296/5572/1466 Combinatorial Synthesis of Genetic Networks] Cabrevelin C. Guet, Michael B. Elowitz, Weihong Hsing, Stanislas Leibler
[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2132448 Programming gene expression with combinatorial promoters] Robert Sidney Cox, III, Michael G Surette, and Michael B Elowitz
Biobricks of Interest:
BBa_K091101 pTet_Lac hybrid promoter
BBa_R0010 lacl regulated promoter
BBa_R0040 TetR repressible promoter
BBa_K137125 LacI-repressed promoter B4
July 10
Today we came up with a schematic of a possible decoder:
We looked more closely at the biobricks: BBa_I14032, BBa_R0040, BBa_K137125, BBa_K091101.
BBa_R0040 and BBa_K091101 were available to us in the registry distribution so we transformed these into cells to test them.
July 13
Today in lab we inoculated a colony of the cells that we transformed the biobricks into yesterday.
Concerns: We want the sRNA gene on a high copy plasmid and the target sequence on low copy plasmid. This could require up to 7 different plasmids to transform into the cell. We need to look into the feasibility of this.
Other things to consider:
- Can we put all of the sRNA expressing genes and their promoters on one plasmid(with all the target sequences and the genes that go with)? would this be too many ligations? How would we tell if they all worked? Run a gel?
- Would we co-transform the plasmids or do them separately?
July 14
In lab we performed a miniprep of the inoculated colony from yesterday to isolate the DNA. It took some time to get access to the centrifuge as it was in use for much of the morning.
At the meeting with the professors they suggested that we take a look at the availabe GFP generator biobricks in the registry. It was also suggested that we would need DH5 alpha Z E. Coli cells if we are to use the lac promoter. It was also mentioned that about two plasmids transformed into a cell was standard and that we could probably get three.
July 15
BBa_I732913 an [aTC] -> RFP measurement biobrick may be useful.
