The Escherichia coli ribosome is a molecular machine capable of sequence-defined polymerization of -amino acids into proteins, a feat unmatched by any other current synthetic catalyst. It is complex in its structure, comprised of 3 RNA parts (the 5S, 16S, and 23S ribosomal RNAs) and 54 ribosomal proteins (r-proteins). Efforts in synthetic biology have focused heavily on engineering the ribosome, and more broadly, the translation apparatus for designer functions, and ample progress has been made in the field of ribosome engineering. However, ribosome engineering is still limited by a lack of methods to engineering large RNA machines as well as efforts to “escape” the evolutionary valley of the ribosome’s fitness landscape. In this work, we present advances made to address these limitations, with particular focus on applying principles rooted in design of molecular machines to the ribosome. The key idea is to free the ribosome from the constraints of its natural function, the synthesis of the cellular proteome. Towards this, the development of the tethered ribosome (termed Ribo-T), in which the small and large subunit rRNAs (16S & 23S) are covalently linked, has been groundbreaking, but is limited by its relatively diminished function compared to a bipartite ribosome. Improvements to Ribo-T function would accelerate ongoing and future efforts to evolve Ribo-T’s catalytic active site and related motifs towards new chemistries beyond peptide bond formation between two -amino acids, opening up the field for the synthesis of new materials and medicines. In this work, I describe the development of Ribo-T v3, which is over 50% improved in orthogonal GFP production and over 96% improved in upkeeping cellular life compared to the previous state-of-the-art. To achieve this, I invented a method termed Evolink, which allows for high throughput directed evolution of multiple coding regions of a molecular machine that may be far apart in primary sequence, but proximal in 3D space and likely interacting. Another way to imagine escaping the ‘evolutionary valley’ of the wild type ribosome sequence is to change the sequence and architecture of the ribosome. I present in this work our efforts to minimize the bacterial ribosome in vitro, which maps permissible regions for deletion. Further, I demonstrate the use of de novo 3D RNA structure prediction algorithms to rescue rRNA deletions that at first glance appear to be fatal to ribosome function. Minimized ribosomes allow the engineer a different starting point for directed evolution as well as reducing the size and complexity of the ribosome. From this work, we hope to one day distill structure-function rules in the bacterial ribosome, leading to elucidations in fundamental ribosome assembly as well as ribosome function. Taken together, I hope that the work presented in this dissertation will enable further acceleration of efforts in ribosome engineering and genetic code expansion to bring forth new classes of sequence-defined polymers with far-reaching applications in medicine, energy, and materials.

Creator

• Kim, Do Soon

DOI

• https://doi.org/10.21985/n2-h2p4-ak75

Subject

• Bioengineering
• Bioinformatics
• Chemical engineering

Language

• en

Alternate Identifier

• etdadmin_upload_831423
• http://dissertations.umi.com/northwestern:15634

Keyword

• Synthetic Biology
• RNA Design
• Ribosome Engineering

Date created

• 2020-01-01

Resource type

• Dissertation

Rights statement

• In Copyright