Engineering multi-signal synthetic biological systems

Synthetic biology involves engineering living cells for applications such as bioremediation, tissue engineering, and biomaterial fabrication, as well as for the purpose of elucidating the underlying principles of natural systems. To date, efforts in this emerging field have focused on programming the behavior of individual cells and on coordinating the activity of multiple cells using a single intercellular signal. Going beyond these efforts by equipping engineered cell populations with the ability to send and receive multiple different signals opens up a wide range of new possibilities. Multiple intercellular signals can be used to divide the tasks of an overall job among different populations, direct complex spatial behavior, and sense and report several environmental conditions. However, engineering multi-signal systems presents many challenges. For example, crosstalk between signaling pathways, sub-optimal signal synthesis and/or detection, and interactions between the signaling pathways and the host's native processes must be addressed.; In this thesis, I discuss successful strategies that I used to introduce new intercellular signaling pathways into cells, optimize the performance of signaling pathway components, and construct synthetic multi-signal systems. I developed a technique for amplifying weak transcriptional responses to intercellular signals, and I employed this technique to identify candidate signal response elements useful for constructing synthetic systems. After building upon these elements and introducing two different communication channels into Escherichia coli cells, I proceeded to implement and test the consensus system, a multi-signal system that coordinates the behavior of two populations of engineered bacteria through bi-directional communication. Since even more complex multi-signal systems may require optimization of signaling components, I successfully enhanced the properties of signal response elements. Finally, to explore approaches for engineering highly complex multi-signal systems, I designed, modeled, and made initial experimental progress towards a system that directs formation of gene expression patterns across a homogeneous lawn of cells. Collectively, this work provides a foundation of genetic building blocks and implementation approaches for engineering complex, multi-signal systems.