A simplified and efficient Agrobacterium tumefaciens electroporation method.
Identifieur interne : 000198 ( Main/Exploration ); précédent : 000197; suivant : 000199A simplified and efficient Agrobacterium tumefaciens electroporation method.
Auteurs : Evelin Kámán-T Th [Hongrie] ; Mikl S Pogány [Hongrie] ; Tamás Dank [Hongrie] ; Ágnes Szatmári [Hongrie] ; Zoltán Bozs [Hongrie]Source :
- 3 Biotech [ 2190-572X ] ; 2018.
Abstract
Agrobacterium tumefaciens is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent Agrobacterium cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g., Agrobacterium strains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly into Agrobacterium omitting the E. coli transformation step and accelerating the cloning procedure further.
DOI: 10.1007/s13205-018-1171-9
PubMed: 29487777
PubMed Central: PMC5821610
Affiliations:
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type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><i>Agrobacterium tumefaciens</i> is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent <i>Agrobacterium</i> cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g., <i>Agrobacterium</i> strains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly into <i>Agrobacterium</i> omitting the <i>E. coli</i> transformation step and accelerating the cloning procedure further.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">29487777</PMID><DateRevised><Year>2020</Year><Month>09</Month><Day>30</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">2190-572X</ISSN><JournalIssue CitedMedium="Print"><Volume>8</Volume><Issue>3</Issue><PubDate><Year>2018</Year><Month>Mar</Month></PubDate></JournalIssue><Title>3 Biotech</Title><ISOAbbreviation>3 Biotech</ISOAbbreviation></Journal><ArticleTitle>A simplified and efficient <i>Agrobacterium tumefaciens</i> electroporation method.</ArticleTitle><Pagination><MedlinePgn>148</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1007/s13205-018-1171-9</ELocationID><Abstract><AbstractText><i>Agrobacterium tumefaciens</i> is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent <i>Agrobacterium</i> cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g., <i>Agrobacterium</i> strains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly into <i>Agrobacterium</i> omitting the <i>E. coli</i> transformation step and accelerating the cloning procedure further.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Kámán-Tóth</LastName><ForeName>Evelin</ForeName><Initials>E</Initials><AffiliationInfo><Affiliation>Department of Pathophysiology, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary.</Affiliation><Identifier Source="ISNI">0000 0001 2149 4407</Identifier><Identifier Source="GRID">grid.5018.c</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Pogány</LastName><ForeName>Miklós</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Department of Pathophysiology, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary.</Affiliation><Identifier Source="ISNI">0000 0001 2149 4407</Identifier><Identifier Source="GRID">grid.5018.c</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Dankó</LastName><ForeName>Tamás</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Department of Pathophysiology, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary.</Affiliation><Identifier Source="ISNI">0000 0001 2149 4407</Identifier><Identifier Source="GRID">grid.5018.c</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Szatmári</LastName><ForeName>Ágnes</ForeName><Initials>Á</Initials><AffiliationInfo><Affiliation>Department of Pathophysiology, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary.</Affiliation><Identifier Source="ISNI">0000 0001 2149 4407</Identifier><Identifier Source="GRID">grid.5018.c</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Bozsó</LastName><ForeName>Zoltán</ForeName><Initials>Z</Initials><Identifier Source="ORCID">0000-0002-4608-775X</Identifier><AffiliationInfo><Affiliation>Department of Pathophysiology, Centre for Agricultural Research, Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary.</Affiliation><Identifier Source="ISNI">0000 0001 2149 4407</Identifier><Identifier Source="GRID">grid.5018.c</Identifier></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D002363">Case Reports</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>02</Month><Day>22</Day></ArticleDate></Article><MedlineJournalInfo><Country>Germany</Country><MedlineTA>3 Biotech</MedlineTA><NlmUniqueID>101565857</NlmUniqueID><ISSNLinking>2190-5738</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Agrobacterium</Keyword><Keyword MajorTopicYN="N">Direct transformation</Keyword><Keyword MajorTopicYN="N">Electroporation</Keyword><Keyword MajorTopicYN="N">Plate method</Keyword></KeywordList><CoiStatement>Compliance with ethical standardsThe authors declare that they have no conflicts of interest in the publication.</CoiStatement></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2017</Year><Month>12</Month><Day>08</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2018</Year><Month>02</Month><Day>14</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2018</Year><Month>3</Month><Day>1</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2018</Year><Month>3</Month><Day>1</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2018</Year><Month>3</Month><Day>1</Day><Hour>6</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">29487777</ArticleId><ArticleId IdType="doi">10.1007/s13205-018-1171-9</ArticleId><ArticleId IdType="pii">1171</ArticleId><ArticleId IdType="pmc">PMC5821610</ArticleId></ArticleIdList><ReferenceList><Reference><Citation>Methods Mol Biol. 1995;55:63-72</Citation><ArticleIdList><ArticleId IdType="pubmed">8528423</ArticleId></ArticleIdList></Reference><Reference><Citation>Plant Physiol. 2008 Feb;146(2):325-32</Citation><ArticleIdList><ArticleId IdType="pubmed">18250230</ArticleId></ArticleIdList></Reference><Reference><Citation>Mol Biotechnol. 1998 Apr;9(2):155-9</Citation><ArticleIdList><ArticleId IdType="pubmed">9658392</ArticleId></ArticleIdList></Reference><Reference><Citation>Plant Physiol. 2003 Nov;133(3):948-55</Citation><ArticleIdList><ArticleId IdType="pubmed">14612581</ArticleId></ArticleIdList></Reference><Reference><Citation>Methods Mol Biol. 2016;1404:581-95</Citation><ArticleIdList><ArticleId 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Agnes" uniqKey="Szatmari A" first="Ágnes" last="Szatmári">Ágnes Szatmári</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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