Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Biochemistry

Supervisor

O'Donoghue, Patrick

Abstract

In Chapter 1, site-specifically phosphorylated variants of the oncogene Akt1 were made in Escherichia coli using the orthogonal translation system that enable genetic code expansion with phosphoserine. The differentially phosphorylated variants of Akt1 were used to validate newly predicted Akt1 substrates. The predicted target sites of the peptide substrates were synthesized and subjected to in vitro kinase assays to quantify the activity of each Akt1 phosphorylated variant towards the predicted peptide. A previously uncharacterized kinase-substrate interaction between Akt1 and a peptide derived from RAB11 Family Interacting Protein 2 (RAB11FIP2) was validated in vitro. Chapter 2 describes the preliminary development of a novel orthogonal translation system for Bacillus subtilis. The work presented outlines the design process: from selection of the components to the generation of an all-in-one plasmid containing the orthogonal translation system. The work demonstrates stable integration of the orthogonal translation system into the B. subtilis genome.

Summary for Lay Audience

Nearly every living organism on the planet studied to date uses the same standard 20 amino acid building blocks to create proteins and enzymes. Each of these 20 amino acids contains a unique chemical functional group, including, for example, acidic/basic groups, aromatic rings, sulfur groups, that each possess distinct chemical properties. Using different combinations and sequences of these 20 building blocks, organisms can produce thousands of different proteins with widely different functions.

Recent advances in genetics and molecular biology have led to the development of technologies that can expand the repertoire of amino acids that are available for organisms beyond these natural 20. The ability to expand the genetic code has led to 100’s of different unnatural or non-standard amino acids being used by a wide range of organisms from bacteria to mice. These new amino acids can revolutionize the types of proteins that organisms can produce by providing them with chemical functional groups that were previously not available. The work presented here makes use of such a system to produce active variants of a human protein called Akt1 that is commonly overactivated in cancers. These active Akt1 variants are made by introducing phosphorylation amino acids into the Akt1 protein, leading to an active Akt1 enzyme. By testing these active variants, we discovered a novel protein targets that Akt1 was able to act on. Results like these enhance our understanding of the scope of Akt1’s role regulating pathways linked to cancer and provide leads to investigate potential new drug targets. In the second chapter, I outline pioneering efforts towards the creation of a novel genetic code expansion technology in a species of bacteria (Bacillus subtilis) that did not previously have such a system. The design process, starting with the careful selection of the components and eventual assembly into a single plasmid DNA and integration into the B. subtilis genome is presented.

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