Rational Design of Genetic Circuits and Biological Modules

Synthetic biology has exploited genetic engineering technology and systems theory to decipher the nature of interactions and apply the knowledge towards design and manufacture of new biological functions. Engineering biological systems with new functions has many promising applications in bioenergy, medicine, and natural computing. In this work, I designed and implemented various biological circuits in microbes, and evaluate functions of the systems both theoretically and experimentally through computational modeling and quantitative measurement of cellular dynamics. In the first chapter of this dissertation, I provide the comprehensive view of synthetic biology explored at three abstraction levels--at metabolite level, gene level, and community member level--and how those levels are interconnected to each other. In the second chapter, the design and implementation of a tunable delay-generating circuit for biocomputing purposes is discussed. Delay is an important building block in biological circuits to construct dynamic regulatory networks such as pulses, oscillations, and time-derivative calculations. At the end of the second chapter, the limitations of single cell engineering are raised, which motivates me to investigate on the resource constraints on the host processes and synthetic pathways in the third chapter. For bioenergy applications, it is desirable to divert energy spent on native or housekeeping gene processes to synthetic pathways to maximize product yield. In our published work, we show that the use of a sequence-dependent endoribonuclease, {\it mazF}, can funnel intracellular resources to a synthetic circuit and increase production of a high-valued metabolite via the programmed degradation of non-circuit mRNA. In the fourth chapter, we analyze intercellular gene regulation in spatial configuration. To validate the theoretical framework, we engineer two strains of cells for a synergistic behavior, and elicit bistable contrasting pattern formation. Synthetic ecology, where cells work in cooperation to achieve more complex social behavior, is critical for advancing the capabilities of synthetic biology to implement an ecological function above the level of one organism. In the final chapter, I develop gene expression tools enabling the use of more diverse microbes. In this project, we create a registry of well-characterized gene regulatory elements for predictable heterologous gene expression in diverse bacterial hosts, including plant pathogens, plant-growth promoting, and bioremediation bacteria, using fluorescence-activated cell sorting and high-throughput sequencing. The large amount of sequencing data also enables statistical modeling of regulatory elements to design a pathway de novo with a set of desired gene expression levels. I believe that the findings described in this dissertation will be useful in advancing various applications of synthetic biology in agriculture, medicine, and bioenergy. For example, the resource allocation by {\it mazF} has further utility in bioenergy production when incorporated in polyclonal or differentiated population. One key challenge in translation of synthetic biology research to real world applications is the high mutation rates and escape mutants that typically result from engineering a large synthetic pathway in a single host. This is due to the selection pressure from high metabolic load. The findings described in this work will help inform engineering of microbial communities to carry out distributed cell behavior, so that each member produces part of the overall pathway and passage intermediates to neighboring members for complete production. This circumvents selection risk while still delivering high product yields. Another compelling application of synthetic biology is to optimize probiotic microbes for production of high-utility metabolites in plant symbiosis and human gut microbiota for applications in agriculture and healthcare. Expansion of gene expression tools and parts development in diverse microbes will accelerate exploitation of the probiotic strains to enhance their beneficial activities in the environment.

Advisor: Arkin, Adam P