Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR

Detalhes bibliográficos
Autor(a) principal: Silva, Filomena
Data de Publicação: 2011
Outros Autores: Lourenço, Olga, Baptista, Cláudio, Queiroz, João, Domingues, F.C.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10400.6/7637
Resumo: DNA-based vaccines and gene therapies have rapidly evolved since the first demonstration of in vivo gene expression by naked DNA injection. When optimizing their production, the major goals in the fermentation process are to maximize the specific plasmid yield, as well as plasmid quality, while minimizing plasmid-mediated metabolic burden. Due to this fact, the development of new and fast monitoring techniques able to easily assess plasmid-mediated metabolic burden and process productivity is in demand. In this work, Escherichia coli DH5 alpha harboring plasmid pVAX1-LacZ was grown in semi-defined media using several pDNA induction strategies. The effect of the induction strategy on cell physiology and overall process productivity was monitored using multi-parameter flow cytometry and real-time qPCR. All induction strategies caused cell filamentation and decreased viability at the end of fermentation. The results also suggest that an amino acid limitation with AMP addition induction strategy resulted in the highest specific yields (20.94 mg/g) and, concomitantly, highest plasmid copy number (1070 per cell). In conclusion, amino acid limitation with AMP addition seems to be a suitable approach to be implemented at a large scale level since it does not require any additional energy and it has proved to be efficient in plasmid amplification.
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spelling Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCRPlasmid DNAAmino acid limitationTemperature up-shiftFlow cytometryReal-time qPCRDNA-based vaccines and gene therapies have rapidly evolved since the first demonstration of in vivo gene expression by naked DNA injection. When optimizing their production, the major goals in the fermentation process are to maximize the specific plasmid yield, as well as plasmid quality, while minimizing plasmid-mediated metabolic burden. Due to this fact, the development of new and fast monitoring techniques able to easily assess plasmid-mediated metabolic burden and process productivity is in demand. In this work, Escherichia coli DH5 alpha harboring plasmid pVAX1-LacZ was grown in semi-defined media using several pDNA induction strategies. The effect of the induction strategy on cell physiology and overall process productivity was monitored using multi-parameter flow cytometry and real-time qPCR. All induction strategies caused cell filamentation and decreased viability at the end of fermentation. The results also suggest that an amino acid limitation with AMP addition induction strategy resulted in the highest specific yields (20.94 mg/g) and, concomitantly, highest plasmid copy number (1070 per cell). In conclusion, amino acid limitation with AMP addition seems to be a suitable approach to be implemented at a large scale level since it does not require any additional energy and it has proved to be efficient in plasmid amplification.uBibliorumSilva, FilomenaLourenço, OlgaBaptista, CláudioQueiroz, JoãoDomingues, F.C.2019-11-28T12:28:35Z20112011-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.6/7637eng10.1016/j.procbio.2010.08.001metadata only accessinfo: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:RCAAP2023-12-15T09:47:08Zoai:ubibliorum.ubi.pt:10400.6/7637Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:48:06.729647Repositório 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 Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
title Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
spellingShingle Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
Silva, Filomena
Plasmid DNA
Amino acid limitation
Temperature up-shift
Flow cytometry
Real-time qPCR
title_short Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
title_full Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
title_fullStr Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
title_full_unstemmed Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
title_sort Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
author Silva, Filomena
author_facet Silva, Filomena
Lourenço, Olga
Baptista, Cláudio
Queiroz, João
Domingues, F.C.
author_role author
author2 Lourenço, Olga
Baptista, Cláudio
Queiroz, João
Domingues, F.C.
author2_role author
author
author
author
dc.contributor.none.fl_str_mv uBibliorum
dc.contributor.author.fl_str_mv Silva, Filomena
Lourenço, Olga
Baptista, Cláudio
Queiroz, João
Domingues, F.C.
dc.subject.por.fl_str_mv Plasmid DNA
Amino acid limitation
Temperature up-shift
Flow cytometry
Real-time qPCR
topic Plasmid DNA
Amino acid limitation
Temperature up-shift
Flow cytometry
Real-time qPCR
description DNA-based vaccines and gene therapies have rapidly evolved since the first demonstration of in vivo gene expression by naked DNA injection. When optimizing their production, the major goals in the fermentation process are to maximize the specific plasmid yield, as well as plasmid quality, while minimizing plasmid-mediated metabolic burden. Due to this fact, the development of new and fast monitoring techniques able to easily assess plasmid-mediated metabolic burden and process productivity is in demand. In this work, Escherichia coli DH5 alpha harboring plasmid pVAX1-LacZ was grown in semi-defined media using several pDNA induction strategies. The effect of the induction strategy on cell physiology and overall process productivity was monitored using multi-parameter flow cytometry and real-time qPCR. All induction strategies caused cell filamentation and decreased viability at the end of fermentation. The results also suggest that an amino acid limitation with AMP addition induction strategy resulted in the highest specific yields (20.94 mg/g) and, concomitantly, highest plasmid copy number (1070 per cell). In conclusion, amino acid limitation with AMP addition seems to be a suitable approach to be implemented at a large scale level since it does not require any additional energy and it has proved to be efficient in plasmid amplification.
publishDate 2011
dc.date.none.fl_str_mv 2011
2011-01-01T00:00:00Z
2019-11-28T12:28:35Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
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dc.identifier.uri.fl_str_mv http://hdl.handle.net/10400.6/7637
url http://hdl.handle.net/10400.6/7637
dc.language.iso.fl_str_mv eng
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dc.relation.none.fl_str_mv 10.1016/j.procbio.2010.08.001
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