Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Yasutaka Kakui, Tomonari Sunaga, Kunio Arai, James Dodgson, Liang Ji, Attila Csikász-Nagy, Rafael Carazo-Salas, Masamitsu Sato

    Research output: Contribution to journalArticle

    10 Citations (Scopus)

    Abstract

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and timeconsuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a 'module', can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.

    Original languageEnglish
    Article number150054
    JournalOpen Biology
    Volume5
    Issue number6
    DOIs
    Publication statusPublished - 2015 Jun 24

    Fingerprint

    Schizosaccharomyces
    Yeast
    Plasmids
    Genes
    Cloning
    Chromosomes
    Organism Cloning
    Gene Knock-In Techniques
    DNA
    DNA Shuffling
    Homologous Recombination
    Libraries
    Proteins
    Research Personnel

    Keywords

    • Chromosomal integration
    • Cloning
    • Fission yeast
    • Fluorescent protein
    • Plasmid

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Neuroscience(all)
    • Immunology

    Cite this

    Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe. / Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu.

    In: Open Biology, Vol. 5, No. 6, 150054, 24.06.2015.

    Research output: Contribution to journalArticle

    Kakui, Y, Sunaga, T, Arai, K, Dodgson, J, Ji, L, Csikász-Nagy, A, Carazo-Salas, R & Sato, M 2015, 'Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe', Open Biology, vol. 5, no. 6, 150054. https://doi.org/10.1098/rsob.150054
    Kakui, Yasutaka ; Sunaga, Tomonari ; Arai, Kunio ; Dodgson, James ; Ji, Liang ; Csikász-Nagy, Attila ; Carazo-Salas, Rafael ; Sato, Masamitsu. / Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe. In: Open Biology. 2015 ; Vol. 5, No. 6.
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