Construction of a fast protein-protein interaction toggle switch in Saccharomyces cerevisiae

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Deepak Mishra, Jim Broach, Ron Weiss

MIT, United States

While protein-protein interactions are ubiquitous in natural biological systems, the use of protein-protein interactions in synthetic biology has been minimal. Several successful strategies have slowly emerged including modular domain recombination and signal pathway diversion for the creation of small unidirectional pathways that ultimately transduce signals to transcription. However, there have not been any complete circuits built that are based solely on protein-protein interactions. Here we describe the design, construction, and testing of circuits in Saccharomyces cerevisiae based solely on protein-protein interactions. We introduce a new design rule, functional protein scaffolding, in combination with existing practices to form a framework for generalized protein-protein circuit engineering. We apply this framework to create a protein-phosphorylation bistable toggle element by rewiring the existing high osmolarity MAPK cascade with exogenous mammalian, plant, and bacterial components. Our results demonstrate that top-down decomposition in conjunction with bottom-up assembly guided by design rules can be applied to protein-protein interaction based circuits. We anticipate our methodology to be a starting point for more sophisticated protein-protein circuits including the use of toggle switch motifs in tandem with logic operations and sensors to create programmable biosensor elements.