System design of artificial genetic circuit by integration of dry and wet experiment

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By using lambda phage promoters, construction of oscillatory genetic circuit was carried out. From the preliminary experiment, the expressional weakness of pRM promoter is problem for the oscillation. In order to increase the expression level from pRM promoter, several mutations of pRM and RBS with higher translation efficiency were applied to this circuit. Despite these efforts, we could not get the continuous oscillation.By using lac and tet repressors, we tried to construct a toggle switch. placIq-lacI gene was introduced into genome, on low copy plasmid, or medium copy plasmid. The toggle switch on high copy plasmid was transformed with each pLlacIq-lacI gene. As the result of the GFP expression experiment, only the strain with low copy plasmid as a source of lac repressor worked as we desired. Mathematical model was also constructed.