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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

14 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

Keywords

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

ASJC Scopus subject areas

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

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