{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/23624"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/23624","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Development and application of a gene transfer system in Clostridium perfringens","abstract":"Initially, electroporation-induced transformation of intact cells of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ with plasmids pAM$\\beta$1 and pHR106 resulted in transformation efficiencies of 1.4 $\\times$ 10$\\sp2$ and 1.2 $\\times$ 10$\\sp3$ transformants/$\\mu$g DNA, respectively. An examination of parameters involved in the electrotransformation process determined that the following factors improved the transformation efficiency of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$: (1) a reduction in cuvette volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 $\\mu$g/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) three-fold concentration of cell density (3.0 $\\times$ 10$\\sp8$ CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the optimized conditions resulted in transformation efficiencies for C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ ranging from 7.1 transformants/$\\mu$g DNA for plasmid plP401 to 9.2 $\\times$ 10$\\sp4$ transformants/$\\mu$g DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 $\\times$ 10$\\sp6$ transformants/$\\mu$g DNA for C. perfringens strain 13. In addition to C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$, eight out of eleven C. perfringens strains examined were transformable using the optimized protocol. Examination of nuclease activity suggested that DNase does not appear to be a factor in the C. perfringens strain-specific electrotransformation protocol.","abstract_html":"Initially, electroporation-induced transformation of intact cells of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ with plasmids pAM<span class=\"etd-inline-math\">&beta;</span>1 and pHR106 resulted in transformation efficiencies of 1.4 $\\times$ 10$\\sp2$ and 1.2 $\\times$ 10$\\sp3$ transformants/<span class=\"etd-inline-math\">&mu;</span>g DNA, respectively. An examination of parameters involved in the electrotransformation process determined that the following factors improved the transformation efficiency of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$: (1) a reduction in cuvette volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 <span class=\"etd-inline-math\">&mu;</span>g/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) three-fold concentration of cell density (3.0 $\\times$ 10$\\sp8$ CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the optimized conditions resulted in transformation efficiencies for C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ ranging from 7.1 transformants/<span class=\"etd-inline-math\">&mu;</span>g DNA for plasmid plP401 to 9.2 $\\times$ 10$\\sp4$ transformants/<span class=\"etd-inline-math\">&mu;</span>g DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 $\\times$ 10$\\sp6$ transformants/<span class=\"etd-inline-math\">&mu;</span>g DNA for C. perfringens strain 13. In addition to C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$, eight out of eleven C. perfringens strains examined were transformable using the optimized protocol. Examination of nuclease activity suggested that DNase does not appear to be a factor in the C. perfringens strain-specific electrotransformation protocol.","abstract_has_math":true,"creators":["Allen, Steven Paul"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Agriculture, Food Science and Technology","degree_department":null,"school":null,"contributors":["Blaschek, Hans-Peter M."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T14:21:01Z","date_published":"2011-05-07T14:21:01Z","updated_at":"2026-07-22T22:25:22Z","subjects":["Agriculture, Food Science and Technology","Biology, Microbiology"],"languages":["eng"],"rights":["Copyright 1990 Allen, Steven Paul"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114159","(UMI)AAI9114159"],"render_values":[{"text":"AAI9114159","href":null,"code":true},{"text":"(UMI)AAI9114159","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/23624","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Blaschek, Hans-Peter M."]},{"key":"dc:creator","label":"Author","values":["Allen, Steven Paul"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T14:21:01Z","10000-01-01","1990"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Agriculture, Food Science and Technology","Biology, Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Agriculture, Food Science and Technology","Biology, Microbiology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1990 Allen, Steven Paul"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114159","(UMI)AAI9114159","http://hdl.handle.net/2142/23624"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Initially, electroporation-induced transformation of intact cells of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ with plasmids pAM$\\beta$1 and pHR106 resulted in transformation efficiencies of 1.4 $\\times$ 10$\\sp2$ and 1.2 $\\times$ 10$\\sp3$ transformants/$\\mu$g DNA, respectively. An examination of parameters involved in the electrotransformation process determined that the following factors improved the transformation efficiency of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$: (1) a reduction in cuvette volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 $\\mu$g/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) three-fold concentration of cell density (3.0 $\\times$ 10$\\sp8$ CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the optimized conditions resulted in transformation efficiencies for C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ ranging from 7.1 transformants/$\\mu$g DNA for plasmid plP401 to 9.2 $\\times$ 10$\\sp4$ transformants/$\\mu$g DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 $\\times$ 10$\\sp6$ transformants/$\\mu$g DNA for C. perfringens strain 13. In addition to C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$, eight out of eleven C. perfringens strains examined were transformable using the optimized protocol. Examination of nuclease activity suggested that DNase does not appear to be a factor in the C. perfringens strain-specific electrotransformation protocol.","The data presented in this thesis represents the first report of the insertion of the streptococcal transposon Tn916 into the C. perfringens chromosome. Introduction of Tn916, harbored on the E. coli plasmid pAM120 delivery vehicle, was accomplished via electrotransformation. The transformation efficiency was 1.2 $\\times$ 10$\\sp2$ Tc$\\sp{\\rm r}$-C. perfringens transformants per $\\mu$g of plasmid DNA. Tn916 has the potential for being exploited as a genetic tool for both insertional mutagenesis and cloning of specific clostridial gene sequences.","This study demonstrated that electroporation-induced transformation is an efficient, reproducible technique for the introduction of plasmid DNA into intact cells of different C. perfringens strains. The introduction of plasmid DNA molecules via this technique should allow for cloning and transposon mutagenesis studies of those C. perfringens genes involved in antibiotic resistance, and bacteriocin and toxin production.","Made available in DSpace on 2011-05-07T14:21:01Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114159.pdf: 4942506 bytes, checksum: 0fbf66f356ffa482b6a30a546331af45 (MD5) Previous issue date: 1990","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T15:05:44Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:31:31-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"]},{"key":"dc:title","label":"Title","values":["Development and application of a gene transfer system in Clostridium perfringens"]}]}],"canonical_facts":{"dc:contributor":["Blaschek, Hans-Peter M."],"dc:creator":["Allen, Steven Paul"],"dc:date":["2011-05-07T14:21:01Z","10000-01-01","1990"],"dc:description":["Initially, electroporation-induced transformation of intact cells of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ with plasmids pAM$\\beta$1 and pHR106 resulted in transformation efficiencies of 1.4 $\\times$ 10$\\sp2$ and 1.2 $\\times$ 10$\\sp3$ transformants/$\\mu$g DNA, respectively. An examination of parameters involved in the electrotransformation process determined that the following factors improved the transformation efficiency of C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$: (1) a reduction in cuvette volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 $\\mu$g/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) three-fold concentration of cell density (3.0 $\\times$ 10$\\sp8$ CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the optimized conditions resulted in transformation efficiencies for C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$ ranging from 7.1 transformants/$\\mu$g DNA for plasmid plP401 to 9.2 $\\times$ 10$\\sp4$ transformants/$\\mu$g DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 $\\times$ 10$\\sp6$ transformants/$\\mu$g DNA for C. perfringens strain 13. In addition to C. perfringens 3624A-Rif$\\sp{\\rm r}$/Str$\\sp{\\rm r}$, eight out of eleven C. perfringens strains examined were transformable using the optimized protocol. Examination of nuclease activity suggested that DNase does not appear to be a factor in the C. perfringens strain-specific electrotransformation protocol.","The data presented in this thesis represents the first report of the insertion of the streptococcal transposon Tn916 into the C. perfringens chromosome. Introduction of Tn916, harbored on the E. coli plasmid pAM120 delivery vehicle, was accomplished via electrotransformation. The transformation efficiency was 1.2 $\\times$ 10$\\sp2$ Tc$\\sp{\\rm r}$-C. perfringens transformants per $\\mu$g of plasmid DNA. Tn916 has the potential for being exploited as a genetic tool for both insertional mutagenesis and cloning of specific clostridial gene sequences.","This study demonstrated that electroporation-induced transformation is an efficient, reproducible technique for the introduction of plasmid DNA into intact cells of different C. perfringens strains. The introduction of plasmid DNA molecules via this technique should allow for cloning and transposon mutagenesis studies of those C. perfringens genes involved in antibiotic resistance, and bacteriocin and toxin production.","Made available in DSpace on 2011-05-07T14:21:01Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114159.pdf: 4942506 bytes, checksum: 0fbf66f356ffa482b6a30a546331af45 (MD5) Previous issue date: 1990","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T15:05:44Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:31:31-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"],"dc:identifier":["AAI9114159","(UMI)AAI9114159","http://hdl.handle.net/2142/23624"],"dc:language":["eng"],"dc:rights":["Copyright 1990 Allen, Steven Paul"],"dc:subject":["Agriculture, Food Science and Technology","Biology, Microbiology"],"dc:title":["Development and application of a gene transfer system in Clostridium perfringens"],"dc:type":["text"],"thesis:degree_discipline":["Agriculture, Food Science and Technology","Biology, Microbiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:22Z"}