A breadboard for synthetic gene networks

Thesis (Ph.D.)--Boston UniversityInterest in the development of increasingly complex synthetic gene networks for the study of natural networks and engineering novel functions has necessitated new approaches to the design and construction of the plasmids upon which they are encoded. Current methods that focus on additive DNA assembly hinder post-construction substitutions. Such modifications are important in facilitating iterative design strategies, which are prevalent due to imperfectly characterized biological components and contexts. We present an approach for the design, construction, and modification of synthetic gene networks termed the Gene Circuit Breadboard. It focuses on the ability to modify constructs to allow for tuning, troubleshooting, or repurposing of networks. We use a specified set of restriction enzymes in conjunction with a library of components that lack those restriction sites to maintain the uniqueness of the sites used for construction. Using this approach, we constructed a genetic toggle switch, added modified ssrA degradation tags to the genes in the toggle, and transformed the toggle into three- and four-node coherent feed-forward loops. The construction and tuning of these networks demonstrated both the need for and performance of our approach, as each circuit required modifications to achieve its intended behavior