Yeast promoter engineering for a genetic oscillator circuit

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Tim Weenink, Tom Ellis

Imperial College London, United Kingdom

Here we show the most recent work towards a minimal degrade-and-fire oscillator in Saccharomyces cerevisiae. This type of oscillator has been shown to work in bacterial chassis, but has not yet been constructed in yeast. Gaining insight in the transition of genetic circuits from one chassis to the next (‘porting’) is important because it expands the applicability and use of work done in one organism to other organisms that have better characteristics in a particular application. The oscillator was chosen as an example circuit because time keeping is one of the basic functions that lie at the foundation of biological computation, along with sensors, switches and memory. The main focus is on the design and characterization of parts. This touches on degradation tags, nuclear localisation tags and promoter repression characteristics in particular. Our mathematical model has shown the need for strong non-linearity in the repression of the promoter as a function of inhibitor concentration. The tetrameric LacI repressor is capable of the intended cooperative effects, however the promoter architecture needs to match this capacity for the intended non-linearity to arise. This imposes specific requirements on the placement of regulatory sequences on the promoter. We show how these design rules resulted in promoter modifications and what the repression characteristics the new promoters have. These characterizations are necessary to determine the most suitable part for the construction of circuits such as a genetic oscillator in yeast, which we are focusing on in particular.