Date of Award

January 2016

Degree Type

Open Access Thesis

Document Type

Master Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

First Advisor

Marcia M. Pierce

Department Affiliation

Biological Sciences

Second Advisor

Rebekah L. Waikel

Department Affiliation

Biological Sciences

Third Advisor

Oliver R. Oakley

Department Affiliation

Biological Sciences

Abstract

The process of using electroporation to introduce plasmid DNA into host cells is a valuable molecular technique that is increasingly employed in labs worldwide. Electroporators are generally small and relatively inexpensive, making them attractive systems to use for a variety of purposes. Electroporation protocols are numerous in the published literature and encompass all cell types, from prokaryotic bacterial cells to eukaryotic human cells. The TargeTron Gene Knockout System by Sigma-Aldrich is an affordable option for the electroporation of numerous bacterial species. However, its use in Listeria monocytogenes has not been extensively characterized. Here we sought to discuss the effectiveness of the TargeTron Gene Knockout System in transforming Listeria monocytogenes via electroporation along with the challenges this process presents.

We attempted to transfect Listeria monocytogenes with two plasmids constructed through SigmaAldrich as part of the TargeTron Gene Knockout System, pACD4K-C and pNL9164, both of which are designed to induce targeted deletion of genes within the host genome. Electroporation was performed under varying conditions, with voltages ranging from 200 to 1250V. Following shock, cells were grown in blood agar or brain heart infusion media containing kanamycin. Pores were induced in the cell wall prior to electroporation by incubating the bacteria in media containing pencillin, ampicillin, or lysozyme. Results show no colonies on selective media post-electroporation for either plasmid across all conditions.

Based on these results, we concluded that this system is not viable for the electroporation of Listeria monocytogenes due to the complicated and expensive techniques required to design appropriate plasmids. However, numerous human errors may have contributed to the lack of success with this system, and therefore, further testing of the protocol with other plasmids should not be ruled out.

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