Studying and Engineering the E. coli 70S Ribosome in vivo and in vitro for the Synthesis of Abiological Polymers

d'Aquino, Anne

doi:https://doi.org/10.21985/n2-xhth-xe58

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Studying and Engineering the E. coli 70S Ribosome in vivo and in vitro for the Synthesis of Abiological Polymers

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The ribosome, the cell’s machine for synthesizing proteins, can be thought of as the chef of the cell. Just as a chef reads a recipe and combines ingredients to create a dish, the ribosome reads cellular instructions and connects building block molecules (amino acids) to construct proteins. Like the final dish that nourishes us, proteins maintain the health of the cell. In culinary arts, a chef’s training dictates their specialty; a chef with basic training prepares traditional staples, while a chef with specialized training masters a specific dish– for example, sushi. This thesis describes how changes (mutations) to the ribosome impact its function and how these mutations can be leveraged to design and build specialized ribosomes. The active site of the ribosome, or the functional heart of the machine, is arguably the most important part of protein synthesis, also known as translation. This site carefully positions amino acid monomers such that they react to form a protein. Studying and mutating the ribosome’s active site has been difficult because changes to the ribosome cause cell sickness and death. This lack of mutational knowledge has hindered the biology and engineering fields and precluded the possibility of leveraging the ribosome for the synthesis of novel polymers. Two key technologies have emerged as a powerful means to design, build, and characterize mutant or engineered ribosomes: cell-free ribosome synthesis, and cellular orthogonal ribosomes. This thesis describes the design, construction, and characterization of ribosomal mutant variants in a cell-free, or in vitro integrated ribosome synthesis, assembly, and translation (iSAT) platform. Using this platform, we have generated sets of fundamental knowledge on the mutational flexibility of the ribosome’s active site and mapped our findings onto the crystal structure to generate structure-function relationships. Furthermore, we have leveraged this platform to rapidly construct and study extended backbone incorporating ribosome constructs. Finally, in an effort to begin studying novel changes to the ribosome in the context of a living cell, efforts to improve an in vivo orthogonal ribosome platform, and use that platform to design and build engineered ribosomes in a cell are described. An overview of the ribosome’s structure, function, and engineering efforts are outlined in Chapter 1. In Chapter 2, a cell-free ribosome synthesis, assembly, and translation (iSAT) platform is leveraged to generate a comprehensive mutational map of the ribosome’s active site. As described in Chapter 3, this system can be used in the construction of multi-mutants for the rapid construction and characterization of ribosomes that incorporate extended backbone monomers. In Chapters 4 and 5, we leverage an in vivo ribosome engineering platform to design, characterize, and use orthogonal tethered ribosomes for expanding biological function. Finally, in Chapter 6, new basic science and synthetic biology questions surrounding the ribosome and translation are explored.

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