Engineering digital, analog and spatiotemporal behavior for synthetic multicellular systems

In this thesis, I establish the design principles for construction of synthetic multi-cellular systems. These will allow researchers to program bacterial cells with the capability to communicate with the environment and other cells and coordinate gene expression of cells temporally and spatially across population. To determine the design principles, I designed and constructed two synthetic multicellular systems that exhibit sophisticated spatiotemporal behavior. The design of these synthetic multicellular systems require engineered intercellular communications and programmed intracellular signal processing mechanisms incorporated in cells.; Both systems consist of engineered sender cells and receiver cells. The first system is the pulse generator in which sender cells communicate to nearby receiver cells. These receiver cells then respond with a transient burst of gene expression whose amplitude and duration depends on the distance between the senders and receivers. The second system is the band detector where receiver cells express an output protein only if the intracellular concentration of signal molecules from sender cells falls within a prespecified and tunable range. This leads to formation of ring like patterns around the sender cells. I demonstrated experimentally how this multicellular system consisting of several different detection thresholds and chemical gradients can generate a variety of interesting spatial patterns such as bullseyes, ellipses, hearts and clovers.; The process of constructing these systems includes integration of synthetic gene networks into two cell populations to create sender and receiver cells. Synthetic gene networks are first designed from well characterized components and tested experimentally. Implementation of synthetic gene networks in cells rarely yield functional circuits on first attempts. Therefore, rational redesign and directed evolution serve as complementary approaches for constructing these synthetic gene networks to elicit desired behavior. The coordinated collective behavior of the engineered cells demonstrates reliable, predictable and sophisticated function despite certain inherent characteristics of cells and substrates. These characteristics include gene expression noise, mutation, cell death, poorly defined and changing extracellular environment. These synthetic multicellular systems will serve as building blocks for a wide range of applications such as programmed tissue engineering, environmental biosensing, and biomaterial fabrication and will provide an improved understanding of naturally occurring biological processes.