The impact of engineered design constraints upon bacteriophage T7 evolution

Since the establishment of molecular biology until the present, a shift has taken place by which engineering of biology has increased in its scope, power, and influence. Synthetic biology is the latest term that advances the overall goal of engineering control of biological systems. Enabled by the recent progress in the ability to read and write nucleic acids, synthetic biology stands out in its desire for standardization of parts, processes, assays, and even organisms. This standardization has led to multiple successful outcomes which justify its utility. However, this standardization via synthetic biology is imbued with an implicit hubris: it may be that certain aspects of biological phenomena are too complex to lend themselves to systemization. Engineering biology, unlike other engineering disciplines, is not obviously governed by a simple series of equations but, rather, united by one universal theme: evolution happens. Because of this, much work has been done in order to study not just how evolution works and but also into controlling, accelerating, and biasing it in a laboratory setting. The former work can be generally classified as “evolutionary biology” and the latter called “directed evolution”. In this dissertation, work is presented which blurs the line between evolutionary biology and directed evolution yielding unanticipated outcomes and new methods of biological control. Three projects are presented in which evolution of bacteriophage T7 is studied in response to rationally engineered design constraints. In the first, the predictability of bacteriophage T7 promoter evolution in response to a novel RNA polymerase is determined. In the second, the evolution of the entirety of the transcriptional apparatus of bacteriophage T7 is studied in the context of an evolutionarily adapted bacteriophage population. Finally, the construction, exploration, and optimization of a designed bacteriophage T7 life-cycling system reliant on proteases are described. Together, this work provides evidence that although the ability to rationally design biology is important, various ways to rationally control and direct evolution offer a complementary strategy to the systematization of synthetic biology.Cellular and Molecular Biolog