Date of Graduation

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor

Robert R. Beitle

Committee Member

Christa Hestekin

Second Committee Member

Ralph Henry

Third Committee Member

Joshua Sakon

Keywords

Biological sciences; Applied sciences; Cloning; Fermentation; Fluorescens; Pseudomonas; Purification; Vaccine

Abstract

In an effort to expand the pool of bacterium useful for biotechnology applications, Pseudomonas fluorescens, a common gram negative microbe, was examined for its ability to function in a recombinant setting. P. fluorescens is ubiquitous in nature and was initially identified as a soil bacterium found in dirt and is typically associated with plant material. Past literature indicates that it shared characteristics common to Escherichia coli and Bacillus subtilis, including simple growth conditions and potential cloning vectors, providing motivation to look into both the upstream and downstream characteristics of this bacterium. First, it was demonstrated that P. fluorescens could be grown to acceptable cell densities in simple batch with cell weights on the order of 60 g/L in the absence of optimization. Lysates of cells were subjected to DEAE ion exchange chromatography to identify the subproteome of soluble proteins which are retained by this resin to guide cellular modifications that reduce the amount and number of host cell proteins (HCPs) encountered during bioseparation. Finally, cloning experiments with Green Fluorescent Protein and FC fragment of tetanus demonstrated both moderate- and large- recombinant DNA products may be obtained from this host.

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