title: Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe.
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 time-consuming, 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.
authors.affiliation: Chromosome Segregation Laboratory, The Francis Crick Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK yasu.kakui@crick.ac.uk.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Kakui
authors.affiliation: Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University TWIns, 2-2 Wakamatsucho, Shinjuku, Tokyo 162-0056, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Sunaga
authors.affiliation: Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University TWIns, 2-2 Wakamatsucho, Shinjuku, Tokyo 162-0056, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Arai
authors.affiliation: The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
authors.forename: James
authors.initials: J
authors.lastname: Dodgson
authors.affiliation: Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University TWIns, 2-2 Wakamatsucho, Shinjuku, Tokyo 162-0056, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Liang
authors.initials: L
authors.lastname: Ji
authors.affiliation: Department of Computational Biology, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige 38010, Italy Randall Division of Cell and Molecular Biophysics and Institute for Mathematical and Molecular Biomedicine, King's College London, London SE1 1UL, UK.
authors.forename: Attila
authors.initials: A
authors.lastname: Csikász-Nagy
authors.affiliation: The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
authors.forename: Rafael
authors.initials: R
authors.lastname: Carazo-Salas
authors.affiliation: Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University TWIns, 2-2 Wakamatsucho, Shinjuku, Tokyo 162-0056, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan masasato@waseda.jp.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Sato
mesh_terms: D015825:Chromosomes, Fungal / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
chemical_list: D004271:DNA, Fungal
keywords: chromosomal integration; cloning; fission yeast; fluorescent protein; plasmid