One of the major goals of the emerging field of synthetic biology is to design synthetic genetic 'circuits' in living cells to program artificial functions. Such programmed cells may serve as efficient cell factories for valuable drugs, chemicals, and biofuels, or intelligent biosensors or therapeutic vehicles that can be implanted in patients. Today, genetic programmers assemble synthetic genetic circuits in a manner similar to how engineers design and build electronic circuits, by connecting a number of 'parts' into a complex ?circuit? synthesized in the form that the cells can interpret (i.e. DNA). However, just as there are many different circuits that can function as a radio, there are many overall circuit designs to achieve a certain cellular function. The main goal of the proposed research is to ask: what is the best strategy to arrive at better (if not the best) circuit designs in the biological context? This is not a trivial question because genetic circuits must function in the complex biological environment. Cells grow, adapt, die, and even mutate while the synthetic circuits run inside them. Ideally, engineered genetic circuits should function robustly in such dynamic and rather unpredictable environment. We propose to adapt the strategy that nature has been using to 'design' its own circuits: evolution. First, we will generate a large number (millions) of different circuits in bacteria through genetic manipulation. The circuits are then subjected to the laboratory evolution process that involves selection and mutation in the controlled environment. We expect that the best circuit designs that robustly function in the living cells emerge from the laboratory evolution process. We will also study how the evolving circuit populations acquire new functions, which is also an important aspect of evolution. These studies are expected to enhance our ability to program living cells with complex and useful functions, as well as to deepen our understanding of how evolution shapes the complex genetic circuits observed in nature.
Broader impacts The tools and insights gained through the proposed research should advance our ability to program living cells for the future practical applications. Genetic parts that are developed through the proposed project will be made available to the research community. Furthermore, the proposed research will actively engage domestic and international undergraduate students as research interns and summer researchers. High school students will also participate in the research through a summer program administered at UC Davis. The proposed project will foster interactions among the young students with diverse backgrounds through cutting-edge research activities.
Intellectual merit: The major intellectulal goal of the project was to develop new genetic methods to control how bacteria such as E. coli behave in laboratory conditions. Such methods and experiments have profound implications in the emerging field of synthetic biology in which researchers attempt to genetically program living cells to perform complex and/or useful tasks, such as synthesizing valuable chemicals from renewable resources. In this project, we have developed a number of potentially useful methods and tools for such applications, for example, small RNA gene switches that can control bacterial gene expression and a "laboratory evolution" strategy that can accelerate genetic programming. These methods have been published and can be used by other researchers in the community to advance the field. Moreover, the research results provide insights into how bacteria use RNA to control gene expression in natural settings. Broader impacts: One of the broader impact outcomes of this project is the dissemination of the research results in the form of publications. In particular, we have published a book chapter that focuses on the technical aspects of our small RNA gene regulators that may be useful for other researchers in the community. Furthermore, the project has provided invaluable training opportunities for young students and researchers, including one underrepresented female graduate student and two female undergraduate students. These students have gained significant research skills which they plan to exploit in their future careers in technology and medicine.
