Design and Optimization of a Genome-Engineering Platform for Systems-Level Optimization of Synthetic Translation Systems

Gowland, Samuel Luke Walker

doi:https://doi.org/10.21985/n2-a08p-0809

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Design and Optimization of a Genome-Engineering Platform for Systems-Level Optimization of Synthetic Translation Systems

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Cellular translation is responsible for the synthesis of proteins, a highly diverse class of macromolecules that form the basis of biological function. In Escherichia coli, harnessing and engineering of the biomolecular components of translation, such as ribosomes, transfer RNAs (tRNAs) and aminoacyl-tRNA synthetases, has led to both biotechnology products (i.e., amylases, insulin) and an expanded genetic code. However, the engineering potential of molecular translation is hampered by the limited capabilities for rapidly sampling the large genomic space necessary to evolve well-coordinated synthetic translation networks inside cells. To address this limitation, I developed a genome engineering method inspired by the action of mobile genetic elements termed mobilization. Mobilization utilizes the stochastic action of the recombinase flippase (FLP) to generate up to ~400 million genomic insertions, deletions, or rearrangements at short flippase recognition target (FRT) sites per mL culture per OD in living E. coli cells. As a model, I applied this approach to evolve faster-growing E. coli strains living exclusively off genomically expressed tethered ribosomes. In an iterative “pulse-passaging scheme,” I generated genomic libraries of cells via induction of FLP recombinase (pulse) followed by passaging the population without induction of FLP to enrich the resulting population for cells with higher fitness. I observed large structural genomic diversity across these cells, with the fastest growing strains exhibiting a 71% increase in growth rate compared to the ancestral strain. I anticipate both these strains, and the mobilization method will be useful tools for synthetic biology efforts to engineer translation systems.

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