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.
Original language | English |
---|---|
Pages (from-to) | 150054 |
Journal | Open Biology |
Volume | 5 |
Issue number | 6 |
DOIs | |
Publication status | Published - Jun 2015 |
Keywords
- Chromosomes, Fungal
- DNA, Fungal
- Genes, Fungal
- Genetic Vectors
- Plasmids
- Promoter Regions, Genetic
- Recombination, Genetic
- Schizosaccharomyces
- Transformation, Genetic
- Journal Article
- Research Support, Non-U.S. Gov't