Rational design and implementation of synthetic genetic digital logic circuits in Saccharomyces cerevisiae
Gander, Miles Winston
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Biology is capable of a wide range of amazing functions, from complex pathway synthesis of high value chemicals to embryonic development. Synthetic biology seeks to harness and control the incredible potential of living systems for the betterment of society. These incredible biological functions are governed by the interaction of complex networks of interacting genes. In principle, the underlying circuitry that control biology function can be recapitulated synthetically, allowing for precise control of biology. In this dissertation, we describe our efforts to rationally design synthetic gene circuits in Saccharomyces cerevisiae. The main thrust of this work is the development, analysis and demonstration of a set of single gene NOR gates based on the CRISPR/Cas9 system. Using these NOR gates we constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Our NOR gates allowed for the construction of the largest eukaryotic gene circuits to date. We detail the process of development of the NOR gates, their limitations and considerations for future use and improvement of the single gene circuit components. The NOR gate technology represents an advancement of the state of the art in synthetic gene circuit design can in principle be used to implement arbitrary internal logic for a variety of synthetic cellular decision making systems, such as those being explored for diagnostics, therapeutics, and development.
- Electrical engineering