Development and Scaling of Differentiation Circuit Architectures for Improving the Evolutionary Stability of Burdensome Functions in E. coli

Citation

Williams, Rory Logan (2022) Development and Scaling of Differentiation Circuit Architectures for Improving the Evolutionary Stability of Burdensome Functions in E. coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5k67-b636. https://resolver.caltech.edu/CaltechTHESIS:01042022-184525578

Abstract

With advances in the sequencing and synthesis of DNA, automation, and computation, we are increasingly able to rapidly and reliably program functions into cells. However, because the functions we engineer cells to perform are often both unnecessary for the cell’s survival and burdensome to cell growth, mutation and natural selection can rapidly lead to loss of function. Though numerous strategies have made headway, improving the evolutionary stability of engineered functions remains a goal of the synthetic biology community. To address this problem generally, we developed a strategy relying on integrase-mediated recombination which allows non-producing progenitor cells to differentiate at a tunable rate, thereby continuously replenishing producer cells expressing the orthogonal T7 RNAP. While this strategy removes selective pressure for mutations inactivating the function of interest in the progenitor cell population, a strategy of terminal differentiation, in which the capacity of differentiated cells to grow is limited, was necessary to prevent the expansion of such mutations in the differentiated cell population. To experimentally implement terminal differentiation, we co-opted the R6K plasmid system, using differentiation to simultaneously activate expression of T7 RNAP, and inactivate expression of π protein (an essential factor for R6K plasmid replication), thereby allowing limitation of differentiated cell growth through antibiotic selection. Critically, we demonstrated computationally that terminal differentiation endows the circuit with robustness to mutations which disrupt T7 RNAP driven expression, and to plasmid instability effects that result in decreased expression. Intuitively and computationally identifying the category of mutations which disrupt the differentiation process as the Achilles's heel of terminal differentiation, we developed a redundant architecture using a novel split-π protein system which required 2 mutations to break the circuit. We experimentally demonstrated a trade-off between rate of production and duration of function as the differentiation rate is tuned, an increased benefit of terminal differentiation with higher-burden expression, and that redundancy improves the evolutionary stability of the terminal differentiation architecture. Specifically we achieve a maximum of ~2.8x (single-cassette terminal differentiation) and ~4.2x (redundant terminal differentiation) the total fluorescent protein production achieved from comparable high-burden naive expression in which all cells inducibly express T7 RNAP. We further demonstrate differentiation can enable the expression of even toxic functions, and develop a terminal differentiation circuit architecture which will allow the degree of redundancy and therefore the evolutionary stability of the architecture to be scaled to arbitrary degrees.

Thesis Files