Practical Applications and Future Directions of Genetic Code Expansion: Validation of Novel Akt1 Substrates and the Design of a Synthetic Auxotroph Strain of B. subtilis
| Autor: | McKenna, McShane M |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: | |
| Zdroj: | Electronic Thesis and Dissertation Repository |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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