Because the number of genes an organism possesses is limited and the number of functions each gene can perform is also limited, coordinated activities of a group of genes are necessary for a cell to execute many complex tasks. However, very little is known about the organization and evolution of such gene networks. In this research, a combined in vivo/in vitro evolution approach will be used to "breed" genetic circuits of various properties. The focus will be on the simplest, and the best-characterized class of genetic circuits, the two-gene "toggle switch". Molecular and evolution experiments will be performed to investigate a number of issues concerning the function and evolution of this system. Specifically, the proposed evolutionary method will be used to generate switches with varying degrees of stability with respect to spontaneous switching by cellular noise. The evolved regulatory sequences will be analyzed to identify features that correlate with different degrees of circuit stability. The robustness of these switches to mutation will also be characterized, and possible relationship between the system's stability to noise and its robustness to mutation will be investigated. Finally, the dynamics of the switch evolution will be probed by sequencing the evolved regions at various intermediate stages of the evolution process. Broader impacts of this research include the training of a new generation of students at the interface and the forefront of biophysics, molecular biology, and evolution, and the development of experimental protocols to evolve designer genetic circuits for a variety of bioengineering and biomedical applications.
