Determination of cell viability of superoxide dismutase knockout Escherichia coli strains using oxidative stress assays

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Christopher Mark Beyer (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://www.wcu.edu/404.asp
Advisor
Lori Seischab

Abstract: Cell viability of superoxide dismutase knockout Escherichia coli strains using oxidative stress assays was determined for three strains: Periplasmic knockout (AS393), cytoplasmic knockout (PN134), and periplasmic/cytoplasmic knockout (AS391). The cytoplasmic or endogenous superoxide was generated using the redox-cycling agent paraquat, while the periplasmic or exogenous superoxide was generated using the xanthine/xanthine oxidase enzyme. It was hypothesized that exogenous superoxide produced in the xanthine/xanthine oxidase assay would have the largest effect on the periplasmic knockout (AS393) while endogenous superoxide produced in the paraquat assay would effect the growth of the cytoplasmic knockouts (AS391, PN134). Cell growth was determined using optical density for the three knockout strains along with four additional strains: ATCC4157, DH10B, MG1655, and W3110. Using total cell counts obtained with a hemacytometer, the relationship between optical density and cell number was determined for all seven strains. Fluorescence assays using the nucleic acid dyes SYTO9 and PI were used to obtain the percentage of live cells for each strain, which was essential for the xanthine/xanthine oxidase assay. Each strain had a different optimal concentration of nucleic acid dyes necessary to obtain the highest fluorescence intensity. Using fluorescence dyes required the investigation of the inner filter effect. It was determined that inner filter effects are present and need to be accounted for. The paraquat assay inhibited the growth of the three knockout strains with the periplasmic knockout (AS393) being more affected than the others. The total time of aeration altered the effectiveness of paraquat. Specifically, as aeration time increased, growth inhibition increased. The xanthine/xanthine oxidase assay showed little to no effect on the cell viability of the knockout strains. Alteration of pH from 7.5 to 6.5 showed similar results. Other alterations to the xanthine/xanthine oxidase assay obtained similar results as well.

Additional Information

Publication
Thesis
Language: English
Date: 2012
Subjects
Superoxide dismutase
Escherichia coli -- Growth
Paraquat
Xanthine oxidase