Repository logo
 
Publication

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

2011Journal article Restricted access
Simple item page
dc.contributor.authorSilva, Filomena
dc.contributor.authorLourenço, Olga
dc.contributor.authorBaptista, Cláudio
dc.contributor.authorQueiroz, João
dc.contributor.authorDomingues, F.C.
dc.date.accessioned2019-11-28T12:28:35Z
dc.date.available2019-11-28T12:28:35Z
dc.date.issued2011
dc.description.abstractDNA-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.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1016/j.procbio.2010.08.001pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.6/7637
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.subjectPlasmid DNApt_PT
dc.subjectAmino acid limitationpt_PT
dc.subjectTemperature up-shiftpt_PT
dc.subjectFlow cytometrypt_PT
dc.subjectReal-time qPCRpt_PT
dc.titleImpact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCRpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage181pt_PT
oaire.citation.issue1pt_PT
oaire.citation.startPage174pt_PT
oaire.citation.titleProcess Biochemistrypt_PT
oaire.citation.volume46pt_PT
person.familyNameLourenço
person.familyNameMaia Baptista
person.familyNameQueiroz
person.familyNameDomingues
person.givenNameOlga
person.givenNameCláudio Jorge
person.givenNameJoão
person.givenNameFernanda
person.identifierhttps://scholar.google.pt/citations?user=hFKOd_QAAAAJ&hl=pt-PT&oi=ao
person.identifier.ciencia-id6618-1C4C-D1B7
person.identifier.ciencia-id5115-FCBD-A31F
person.identifier.ciencia-id931E-B66D-E341
person.identifier.ciencia-id6514-0498-3E8F
person.identifier.orcid0000-0002-8401-5976
person.identifier.orcid0000-0002-5658-5445
person.identifier.orcid0000-0002-3096-8325
person.identifier.orcid0000-0002-9540-0853
person.identifier.ridL-3104-2014
person.identifier.ridN-7295-2013
person.identifier.scopus-author-id23097426600
person.identifier.scopus-author-id7003705645
person.identifier.scopus-author-id10139481400
rcaap.embargofctCopyright cedido à editora no momento da publicação.pt_PT
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublicationc890c74a-abc1-4910-aa70-f0da7b4ad902
relation.isAuthorOfPublication1080c532-c737-4b60-bc44-1d18e3c877d7
relation.isAuthorOfPublicationc798c033-c160-4f99-a8ad-04416923943f
relation.isAuthorOfPublication8d5fe6e2-21af-4557-9c46-84103254a18a
relation.isAuthorOfPublication.latestForDiscoveryc890c74a-abc1-4910-aa70-f0da7b4ad902

Files

Original bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR.pdf
Size:
679.63 KB
Format:
Adobe Portable Document Format
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.71 KB
Format:
Item-specific license agreed upon to submission
Description:
Download

Collections

FCS - DCM | Documentos por Auto-Depósito
ICI - CICS | Documentos por Auto-Depósito