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

*この研究の対応する著者

研究成果: Article査読

16 被引用数 (Scopus)

抄録

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.

本文言語English
論文番号150054
ジャーナルOpen Biology
5
6
DOI
出版ステータスPublished - 2015 6月 24

ASJC Scopus subject areas

  • 神経科学(全般)
  • 免疫学
  • 生化学、遺伝学、分子生物学(全般)

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