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.
","abstract_html":"<p>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. </p> <p>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. </p> <p>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. </p>","abstract_has_math":false,"creators":["Frazier, Hilaree Noele"],"institution":"Eastern Kentucky University","degree_name":"Master of Science (MS)","degree_level":"Master's","degree_discipline":"Biological Sciences","degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2016,"date_issued":"2016-01-01T08:00:00Z","date_published":"2016-01-01T08:00:00Z","updated_at":"2026-07-24T02:15:32Z","subjects":["bacteria","electroporation","listeria","plasmid","Microbiology","Molecular Biology"],"languages":[],"rights":["Copyright 2016 Hilaree Noele Frazier"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://encompass.eku.edu/etd/365","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Frazier, Hilaree Noele"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:publisher","label":"Institution","values":["Encompass Digital Archive, Eastern Kentucky University"]},{"key":"dc:type","label":"Dc Type","values":["Master Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biological Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Master's"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (MS)"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Eastern Kentucky University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["bacteria","electroporation","listeria","plasmid","Microbiology","Molecular Biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2016 Hilaree Noele Frazier"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://encompass.eku.edu/etd/365"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:source","label":"Dc Source","values":["Encompass Digital Archive: Online Theses and Dissertations"]},{"key":"dc:title","label":"Title","values":["Efficiency of the TargeTron Gene Knockout System as a Transformative Protocol for the Mutagenesis of Listeria monocytogenes"]}]}],"canonical_facts":{"dc:creator":["Frazier, Hilaree Noele"],"dc:description.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.
"],"dc:format":["application/pdf"],"dc:identifier":["https://encompass.eku.edu/etd/365"],"dc:publisher":["Encompass Digital Archive, Eastern Kentucky University"],"dc:rights":["Copyright 2016 Hilaree Noele Frazier"],"dc:source":["Encompass Digital Archive: Online Theses and Dissertations"],"dc:subject":["bacteria","electroporation","listeria","plasmid","Microbiology","Molecular Biology"],"dc:title":["Efficiency of the TargeTron Gene Knockout System as a Transformative Protocol for the Mutagenesis of Listeria monocytogenes"],"dc:type":["Master Thesis"],"thesis:degree_discipline":["Biological Sciences"],"thesis:degree_level":["Master's"],"thesis:degree_name":["Master of Science (MS)"],"thesis:institution_name":["Eastern Kentucky University"]},"updated_at":"2026-07-24T02:15:32Z"}