Building Platforms to Genetically Encode New Chemistry

• Main Author: Johnson, Alexander M.
• Format: Others
• Language: English
• Published: Boston College 2017
• Subjects: Synthetase Evolution; Unnatural Amino Acid; Yeast Selection
• Online Access: http://hdl.handle.net/2345/bc-ir:107292

Thesis advisor: Abhishek Chatterjee === Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. === Thesis (MS) — Boston College, 2017. === Submitted to: Boston College. Graduate School of Arts and Sciences. === Discipline: Chemistry.