Date of Award

Spring 1-1-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Anushree Chatterjee

Second Advisor

Ryan T. Gill

Third Advisor

Michael R. Shirts

Fourth Advisor

Amy Palmer

Fifth Advisor

Robert Batey

Abstract

Nature is rarely static. Organisms live in diverse and stressful environments that necessitate rapid response strategies for survival. Microorganisms have responded to this by evolving bet-hedging, wherein they exhibit constitutively heterogeneous gene expression to maximize fitness across numerous background. The goal of this thesis is to “hijack” this phenomenon using novel gene expression engineering techniques to alter how bacteria respond to their environments, in order to address pressing societal concerns.

This begins with a systematic exploration of how bacterial gene expression naturally responds to antibiotics and biofuels. This reveals promising gene candidates for targeted manipulation, for which a library of CRISPR gene expression perturbation devices is constructed. This library is applied to Escherichia coli during exposure to antibiotics and biofuels, and the impact of CRISPR perturbation on growth and fitness is quantified. Many perturbations show significant non-heritable improvements or detriments on growth, indicating the potential of this approach for biofuel and antibiotic applications. To improve the desired bacterial response, individual perturbations are combined in a multiplexed fashion. A significant trend towards lower fitness as more perturbations are combined emerges, which is supported by a systematic exploration of combinatorial perturbation libraries. This trend is correlated to a diminished adaptive potential, suggesting the applicability of multiplexed perturbations for restricting bacterial evolution. In a similar vein, the ability of gene expression perturbations to synergize with antibiotics is explored to identify novel potentiating therapies. Significant gene-drug synergies are characterized and used to potentiate antibiotic treatment in an infection model. Therapeutic peptide nucleic acid molecules are subsequently designed to re-sensitize clinically isolated multidrug-resistant bacteria to treatment. Finally, this thesis expands our available synthetic biology toolkit for manipulating gene expression by outlining novel CRISPR engineering strategies. Deactivated CRISPR proteins are fused with bacterial initiation factor one, and the potential for these constructs to increase translation rates by promoting 30S subunit binding to the ribosome is explored. The design of a smart antibiotic utilizing a CRISPR-holin RNA-based kill switch is also presented. Collectively, this thesis demonstrates the power that manipulating gene expression has in affecting desired phenotypes in bacteria.

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