Investigations into the Molecular Mechanisms of Bacterial Pathogen-Host Interactions: Construction of a Dual Plasmid System for Incorporation of Unnatural Amino Acids into Pseudomonas syringae pv. tomato DC3000

Authors

Scotty D. Raber, University of Nebraska-LincolnFollow

Date of this Version

Spring 5-1-2015

Citation

Raber, Scott (2015), Investigations into the Molecular Mechanisms of Bacterial Pathogen-Host Interactions: Construction of a Dual Plasmid System for Incorporation of Unnatural Amino Acids into Pseudomonas syringae pv. tomato DC3000, MS thesis, University of Nebraska

Comments

A Thesis Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemistry, Under the Supervision of Professor Jiantao Guo. Lincoln, Nebraska: May, 2015

Copyright (c) 2015 Scott D. Raber

Abstract

A dual plasmid system for the incorporation of unnatural amino acids into plant pathogen, Pseudomonas syringae pv. tomato DC3000, has been designed. This invention is expected to allow (a) mutations of proteins synthesized by the bacterium, P. syringae pv. tomato DC3000, that can capture molecular targets, especially for such modified proteins secreted by the phytopathogen into the host plant cells of A. thaliana and S. lycopersicum, (b) expression of biological probes in the bacterial species to monitor changes in redox, nutritional, and other small molecule states over pre-, post- and in situ disease stages, and (c) secretion of such biological probes into an infection host organism to monitor intracellular changes of redox, nutritional, and small molecule states in infected host. This plasmid system will allow proteins to feature unnatural amino acids in substitution for selected original residues by site-specific alteration of the protein sequence to the TAG stop codon for recognition exogenous and orthogonal TAG-suppressing tRNA that has been charged with the unnatural amino acid by its cognate tRNA synthetase (RS). Because the suppressor tRNA and its counterpart synthetase are designed to only interact with each other, proteins expressed by the bacterial species containing this orthogonal tRNA/RS pair will enable in vivo production of proteins featuring specialized disease-based monitoring capabilities. In addition, this plasmid system is expected to be compatible not only with other pathovars of the P. syringae genus, but also other phytopathogens and soil bacteria, thereby significantly enhancing the researcher’s ability to understand the role and response of these microorganisms within their environment.

Advisor: Jiantao Guo