Evaluation of a new method for genetic modification of microorganisms

Detalhes bibliográficos
Autor(a) principal: Kwiatkowska, Katarzyna
Data de Publicação: 2011
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10773/7691
Resumo: Nowadays recombinant DNA technology is broadly present in our lives, bringing both hopes and fears. Transformation, even if achievable by several techniques, still faces with unsatisfying efficiencies and lack of universality. In this work, an attempt to evaluate the potential of a new transformation methodology was made. The idea for this work came from food preservation where high hydrostatic pressure is utilized to inactivate microbial flora, mainly by destructive effects on membranes, as for instance cavities. This way, it is rational to think that sub-lethally affected cells would create pores allowing the uptake of DNA. Thus, in order to compel the method to work, a compromise between enough stress and maintenance of some cells still viable and capable to recover must be looked for. In this work, the assessment was performed on bacterial host - Escherichia coli TOP10 and small circular plasmid – pUC19 that provides resistance to ampicillin as a selection marker. Electroporation, the reference technique, resulted in transformation efficiency rates of and transformants/μg of DNA when 0,01 μg/mL plasmid was applied and of and transformants/μg of DNA with 100 μg/mL plasmid (double values are derived from assays carried at distinct days). Concerning pressure, treatments were limited to 50-400 MPa and the time ranged from 10 seconds to 5 minutes. Pressurizations included also examination of two values of compression rate: 5 and 10 MPa/sec and two cycle variants: single and triple. Treatments under 50 and 100 MPa during 2,5 and 5 min, as well as pressurization under 200, 300 and 400 MPa during 1 and 5 min, caused reduction of viability ≥99,99% of initial bacterial population. As a result, values of the potential stress factors were narrowed to 50–200 MPa lasting up to 1 minute at 5 MPa/sec compression rate, with the reduction of viability approximately 90%. Longer treatments were performed with multiple-cycles resulting in number of survivors of approximately 3% higher values (for cells pressurized without plasmid). It was concluded that E. coli may require some time to recover and so rich nutritionally broth should not be added immediately after pressurization, although fluctuations were not of great significance. In majority of the cases, addition of plasmid reduced the number of detected survivors but the variations were almost unnoticeable being in the range of few percent. Some of the experiments resulted in surprising but evident increase of cell number, reaching even up to 7,5-fold increase for the treatment under 100 MPa during 30 sec at normal compression rate with addition of pUC19. Such observations were supposed to origin from disaggregating or germination-inducing activity of pressure. With the results obtained, no successful transformation was observed. Since there exist several potential improvements of methodology (i.e. various host-plasmid sets, induction of cell competences, forms and sizes of DNA) future evaluation could hopefully bring positive results.
id RCAP_3d67c89ea3dbdb94c243784d231d42c5
oai_identifier_str oai:ria.ua.pt:10773/7691
network_acronym_str RCAP
network_name_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository_id_str 7160
spelling Evaluation of a new method for genetic modification of microorganismsBiotecnologiaBactérias patogénicasPlasmídeosAlterações genéticas - MicroorganismosConservação dos alimentosNowadays recombinant DNA technology is broadly present in our lives, bringing both hopes and fears. Transformation, even if achievable by several techniques, still faces with unsatisfying efficiencies and lack of universality. In this work, an attempt to evaluate the potential of a new transformation methodology was made. The idea for this work came from food preservation where high hydrostatic pressure is utilized to inactivate microbial flora, mainly by destructive effects on membranes, as for instance cavities. This way, it is rational to think that sub-lethally affected cells would create pores allowing the uptake of DNA. Thus, in order to compel the method to work, a compromise between enough stress and maintenance of some cells still viable and capable to recover must be looked for. In this work, the assessment was performed on bacterial host - Escherichia coli TOP10 and small circular plasmid – pUC19 that provides resistance to ampicillin as a selection marker. Electroporation, the reference technique, resulted in transformation efficiency rates of and transformants/μg of DNA when 0,01 μg/mL plasmid was applied and of and transformants/μg of DNA with 100 μg/mL plasmid (double values are derived from assays carried at distinct days). Concerning pressure, treatments were limited to 50-400 MPa and the time ranged from 10 seconds to 5 minutes. Pressurizations included also examination of two values of compression rate: 5 and 10 MPa/sec and two cycle variants: single and triple. Treatments under 50 and 100 MPa during 2,5 and 5 min, as well as pressurization under 200, 300 and 400 MPa during 1 and 5 min, caused reduction of viability ≥99,99% of initial bacterial population. As a result, values of the potential stress factors were narrowed to 50–200 MPa lasting up to 1 minute at 5 MPa/sec compression rate, with the reduction of viability approximately 90%. Longer treatments were performed with multiple-cycles resulting in number of survivors of approximately 3% higher values (for cells pressurized without plasmid). It was concluded that E. coli may require some time to recover and so rich nutritionally broth should not be added immediately after pressurization, although fluctuations were not of great significance. In majority of the cases, addition of plasmid reduced the number of detected survivors but the variations were almost unnoticeable being in the range of few percent. Some of the experiments resulted in surprising but evident increase of cell number, reaching even up to 7,5-fold increase for the treatment under 100 MPa during 30 sec at normal compression rate with addition of pUC19. Such observations were supposed to origin from disaggregating or germination-inducing activity of pressure. With the results obtained, no successful transformation was observed. Since there exist several potential improvements of methodology (i.e. various host-plasmid sets, induction of cell competences, forms and sizes of DNA) future evaluation could hopefully bring positive results.Actualmente, a tecnologia de DNA recombinante está presente de modo substancial nas nossas vidas, levando as suas possíveis aplicações simultaneamente a esperança e o medo. A transformação genética, mesmo que realizável com várias técnicas, enfrenta ainda eficiências insatisfatórias em muitos casos e impossibilidade noutros. Neste trabalho, foi realizada uma tentativa para estudar o potencial de um novo método de transformação genética, concretamente de introdução do material genético na célula a transformar. A ideia para este trabalho foi originada da área de conservação dos alimentos, onde a alta pressão hidrostática é utilizada para inactivar microrganismos, principalmente pelos seus efeitos destrutivos sobre as membranas, por exemplo produzindo poros. Deste modo, é razoável supor que nas células stressadas sub-letalmente se criem poros permitindo a introdução de DNA. Assim, para que esta metodologia funcione, um compromisso entre stress suficiente e a manutenção da viabilidade das células deve ser utilizado. Neste trabalho, a avaliação efectuou-se com o hospedeiro bacteriano - Escherichia coli TOP10 e com um pequeno plasmídeo circular – pUC19, que confere uma resistência à ampicilina como marcador. A electroporação, usada como técnica da referência, resultou em taxas de eficiência de (9,47±2,00)×107 e (6,30±0,83)×107 transformantes/μg de DNA quando foi aplicada concentração de plasmídeo 0,01 μg/mL e de (1,18±0,37)×107 e (3,44±0,56)×107 transformantes/μg com 100 μg/mL de plasmídeo. Relativamente à pressão, os tratamentos situaram-se entre 50–400 MPa e o tempo variou dos 10 segundos até aos 5 minutos. Além disso, as pressurizações incluíram um estudo de dois valores da taxa de compressão: 5 e 10 MPa/seg e dois tipos de ciclos: singulares e triplos. Os tratamentos sob 50 e 100 MPa durante 2,5 e 5 min, assim como as pressurizações sob 200, 300 e 400 MPa durante 1 e 5 min, causaram uma redução da viabilidade ≥99,99% da população bacteriana inicial. Portanto, os valores dos potenciais factores de stress foram limitados a 50–200 MPa, até 1 minuto com uma taxa da compressão 5 MPa/seg, com a redução da viabilidade de aproximadamente 90%. Os tratamentos mais longos realizaram-se com os ciclos múltiplos resultando num número de sobreviventes aproximadamente 3% mais alto, para células pressurizadas sem o plasmídeo. Concluiu-se que E. coli pode precisar de algum tempo para recuperar, pelo que é preferível adicionar o meio enriquecido não imediatamente depois uma pressurização, embora as variações não sejam muito significativas. Na maioria dos casos, a adição de plasmídeo reduziu o número dos sobreviventes detectados. Algumas das experiências resultaram num inesperado mas evidente aumento do número das células, até valores 7,5 vezes mais altos para o tratamento sob 100 MPa durante 30 seg com uma taxa da compressão normal e com adição de pUC19. Estas observações podem dever-se à possibilidade da pressão poder causar desagregação de células ou a indução de germinação. Com os resultados obtidos não se observou transformação genética com sucesso. Como potencialmente existem vários parâmetros da metodologia (p. ex. vários conjuntos hospedeiro - plasmídeo, indução das competências celulares, as formas e tamanhos de DNA), uma nova futura avaliação da alta pressão para modificação genética deve ser tentada.Universidade de Aveiro2013-11-27T08:46:03Z2011-07-05T00:00:00Z2011-07-05info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/7691engKwiatkowska, Katarzynainfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T11:13:24Zoai:ria.ua.pt:10773/7691Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:45:19.549933Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Evaluation of a new method for genetic modification of microorganisms
title Evaluation of a new method for genetic modification of microorganisms
spellingShingle Evaluation of a new method for genetic modification of microorganisms
Kwiatkowska, Katarzyna
Biotecnologia
Bactérias patogénicas
Plasmídeos
Alterações genéticas - Microorganismos
Conservação dos alimentos
title_short Evaluation of a new method for genetic modification of microorganisms
title_full Evaluation of a new method for genetic modification of microorganisms
title_fullStr Evaluation of a new method for genetic modification of microorganisms
title_full_unstemmed Evaluation of a new method for genetic modification of microorganisms
title_sort Evaluation of a new method for genetic modification of microorganisms
author Kwiatkowska, Katarzyna
author_facet Kwiatkowska, Katarzyna
author_role author
dc.contributor.author.fl_str_mv Kwiatkowska, Katarzyna
dc.subject.por.fl_str_mv Biotecnologia
Bactérias patogénicas
Plasmídeos
Alterações genéticas - Microorganismos
Conservação dos alimentos
topic Biotecnologia
Bactérias patogénicas
Plasmídeos
Alterações genéticas - Microorganismos
Conservação dos alimentos
description Nowadays recombinant DNA technology is broadly present in our lives, bringing both hopes and fears. Transformation, even if achievable by several techniques, still faces with unsatisfying efficiencies and lack of universality. In this work, an attempt to evaluate the potential of a new transformation methodology was made. The idea for this work came from food preservation where high hydrostatic pressure is utilized to inactivate microbial flora, mainly by destructive effects on membranes, as for instance cavities. This way, it is rational to think that sub-lethally affected cells would create pores allowing the uptake of DNA. Thus, in order to compel the method to work, a compromise between enough stress and maintenance of some cells still viable and capable to recover must be looked for. In this work, the assessment was performed on bacterial host - Escherichia coli TOP10 and small circular plasmid – pUC19 that provides resistance to ampicillin as a selection marker. Electroporation, the reference technique, resulted in transformation efficiency rates of and transformants/μg of DNA when 0,01 μg/mL plasmid was applied and of and transformants/μg of DNA with 100 μg/mL plasmid (double values are derived from assays carried at distinct days). Concerning pressure, treatments were limited to 50-400 MPa and the time ranged from 10 seconds to 5 minutes. Pressurizations included also examination of two values of compression rate: 5 and 10 MPa/sec and two cycle variants: single and triple. Treatments under 50 and 100 MPa during 2,5 and 5 min, as well as pressurization under 200, 300 and 400 MPa during 1 and 5 min, caused reduction of viability ≥99,99% of initial bacterial population. As a result, values of the potential stress factors were narrowed to 50–200 MPa lasting up to 1 minute at 5 MPa/sec compression rate, with the reduction of viability approximately 90%. Longer treatments were performed with multiple-cycles resulting in number of survivors of approximately 3% higher values (for cells pressurized without plasmid). It was concluded that E. coli may require some time to recover and so rich nutritionally broth should not be added immediately after pressurization, although fluctuations were not of great significance. In majority of the cases, addition of plasmid reduced the number of detected survivors but the variations were almost unnoticeable being in the range of few percent. Some of the experiments resulted in surprising but evident increase of cell number, reaching even up to 7,5-fold increase for the treatment under 100 MPa during 30 sec at normal compression rate with addition of pUC19. Such observations were supposed to origin from disaggregating or germination-inducing activity of pressure. With the results obtained, no successful transformation was observed. Since there exist several potential improvements of methodology (i.e. various host-plasmid sets, induction of cell competences, forms and sizes of DNA) future evaluation could hopefully bring positive results.
publishDate 2011
dc.date.none.fl_str_mv 2011-07-05T00:00:00Z
2011-07-05
2013-11-27T08:46:03Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10773/7691
url http://hdl.handle.net/10773/7691
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade de Aveiro
publisher.none.fl_str_mv Universidade de Aveiro
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
_version_ 1799137505131364352