Recombineering reagents for improved inducible expression and selection marker re-use in Schizosaccharomyces pombe.
The fission yeast Schizosaccharomyces pombe is an excellent model organism for cell biology. However, its genetic toolbox is less developed than that of Saccharomyces cerevisiae. In the first part of this study we describe an improved inducible expression vector based on tetracycline regulation of the CaMV35S promoter, which is also capable of chromosomal integration and therefore works in minimal and in rich media. We found that anhydrotetracycline is a superior ligand for induction. Maximum expression levels were observed after 12 h in minimal media (EMM) and after 9 h in rich media (YES), which is faster than the nmt1 promoter system. The system was combined with a convenient recombineering-based subcloning strategy for ease of cloning. In the second part we present four template plasmids, pSVEM-bsd, pSVEM-nat, pSVEM-kan and pSVEM-hph, which harbour four recyclable disruption cassettes based on the Cre recombinase lox71/66 strategy for use in PCR targeting methods. Cre-mediated excision leaves a non-functional mutant lox site in the genome, allowing the reiterative usage of these cassettes for multiple targetings. These cassettes are also configured with dual eukaryotic/prokaryotic promoters so that they can be used for recombineering in E. coli. Amongst other purposes, this permits the rapid and convenient creation of targeting constructs with much longer homology arms for difficult and complex targetings in the Sz. pombe genome.
Erler
Axel
A
Technical University Dresden, Biotec, Department of Genomics, Tatzberg 47-51, 01307 Dresden, Germany.
Maresca
Marcello
M
Fu
Jun
J
Stewart
A Francis
AF
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics; D013754:Tetracyclines / Q000494:pharmacology
C093953:NMT1 protein, S pombe; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D013754:Tetracyclines; C016229:4-epianhydrotetracycline