A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum

No Thumbnail Available
Files
There are no files associated with this item.
Date
2019
Editors
Contact
Journal ISSN
Electronic ISSN
ISBN
Bibliographical data
Publisher
Series
URI (citable link)
DOI (citable link)
ArXiv-ID
International patent number
Link to the license
oops
EU project number
Project
Open Access publication
Restricted until
Title in another language
Research Projects
Organizational Units
Journal Issue
Publication type
Journal article
Publication status
Published
Published in
Algal Research ; 39 (2019). - 101469. - ISSN 2211-9264
Abstract
The recent availability of genome editing tools like TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) for the diatom Phaeodactylum tricornutum has dramatically increased the options to explore diatom biology via reverse genetics. In order to verify that an observed phenotype indeed is directly related to a specific gene knockout and not due to a secondary effect, complementation of the inactivated gene with the wildtype gene and restoration of the wild type phenotype is an essential tool in molecular biology. So far, no strategy for a complementation method has been published for P. tricornutum. Here we demonstrate, as a proof-of principle, the complementation of P. tricornutum AUREO1a knockout strains previously created by TALEN technology. These strains are deficient in the PtAureo1a gene, which is encoding a blue-light dependent transcription factor. pPTbsr, a modified pPha-T1 vector with an antibiotic resistance cassette against Blasticidin served as a complementation vector. In order to avoid the modification of the complementing gene via the potentially still active TALEN nucleases, we have modified the TALEN binding sites of the complementing PtAureo1a gene using synonymous codons. The altered PtAureo1a gene along with its native promoter and terminator was transformed by particle gun bombardment into PtAUREO1a TALEN knockout strains of P. tricornutum. The integration and the expression of PtAUREO1a was confirmed by PCR and western blotting. Due to random integration within the genome, the expression level of the complemented gene may be variable in different lines. Physiological parameters indicated the successful rescue of the wild type phenotype in several lines that showed a similar PtAUREO1a protein content as wild type cells. Our method provides a rapid and efficient tool to complement knockout lines generated by genome editing approaches in P. tricornutum.
Summary in another language
Subject (DDC)
570 Biosciences, Biology
Keywords
Phaeodactylum tricornutum, Complementation, PtAureo1a TALEN based knockout
Conference
Review
undefined / . - undefined, undefined. - (undefined; undefined)
Cite This
ISO 690MADHURI, Shvaita, Carolina RÍO BÁRTULOS, Manuel SERIF, Bernard LEPETIT, Peter G. KROTH, 2019. A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum. In: Algal Research. 39, 101469. ISSN 2211-9264. Available under: doi: 10.1016/j.algal.2019.101469
BibTex
@article{Madhuri2019-05strat-45530,
  year={2019},
  doi={10.1016/j.algal.2019.101469},
  title={A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum},
  volume={39},
  issn={2211-9264},
  journal={Algal Research},
  author={Madhuri, Shvaita and Río Bártulos, Carolina and Serif, Manuel and Lepetit, Bernard and Kroth, Peter G.},
  note={Article Number: 101469}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/45530">
    <dc:contributor>Serif, Manuel</dc:contributor>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/52"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/52"/>
    <dc:contributor>Lepetit, Bernard</dc:contributor>
    <dc:contributor>Río Bártulos, Carolina</dc:contributor>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-03-20T14:24:57Z</dcterms:available>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-03-20T14:24:57Z</dc:date>
    <dc:creator>Madhuri, Shvaita</dc:creator>
    <dc:creator>Lepetit, Bernard</dc:creator>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dc:contributor>Madhuri, Shvaita</dc:contributor>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/45530"/>
    <dc:creator>Serif, Manuel</dc:creator>
    <dc:contributor>Kroth, Peter G.</dc:contributor>
    <dc:creator>Río Bártulos, Carolina</dc:creator>
    <dc:creator>Kroth, Peter G.</dc:creator>
    <dc:language>eng</dc:language>
    <dcterms:issued>2019-05</dcterms:issued>
    <dcterms:title>A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum</dcterms:title>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dcterms:abstract xml:lang="eng">The recent availability of genome editing tools like TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) for the diatom Phaeodactylum tricornutum has dramatically increased the options to explore diatom biology via reverse genetics. In order to verify that an observed phenotype indeed is directly related to a specific gene knockout and not due to a secondary effect, complementation of the inactivated gene with the wildtype gene and restoration of the wild type phenotype is an essential tool in molecular biology. So far, no strategy for a complementation method has been published for P. tricornutum. Here we demonstrate, as a proof-of principle, the complementation of P. tricornutum AUREO1a knockout strains previously created by TALEN technology. These strains are deficient in the PtAureo1a gene, which is encoding a blue-light dependent transcription factor. pPTbsr, a modified pPha-T1 vector with an antibiotic resistance cassette against Blasticidin served as a complementation vector. In order to avoid the modification of the complementing gene via the potentially still active TALEN nucleases, we have modified the TALEN binding sites of the complementing PtAureo1a gene using synonymous codons. The altered PtAureo1a gene along with its native promoter and terminator was transformed by particle gun bombardment into PtAUREO1a TALEN knockout strains of P. tricornutum. The integration and the expression of PtAUREO1a was confirmed by PCR and western blotting. Due to random integration within the genome, the expression level of the complemented gene may be variable in different lines. Physiological parameters indicated the successful rescue of the wild type phenotype in several lines that showed a similar PtAUREO1a protein content as wild type cells. Our method provides a rapid and efficient tool to complement knockout lines generated by genome editing approaches in P. tricornutum.</dcterms:abstract>
  </rdf:Description>
</rdf:RDF>
Internal note
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Contact
URL of original publication
Test date of URL
Examination date of dissertation
Method of financing
Comment on publication
Alliance license
Corresponding Authors der Uni Konstanz vorhanden
International Co-Authors
Bibliography of Konstanz
Yes
Refereed
Unknown