Preparing libraries in Saccharomyces cerevisiae.

Abstract

AbstractHigh recombination frequency and ease of manipulation made the budding yeast, Saccharomyces cerevisiae, the model eukaryotic organism for studies on homologous recombination (1,2). Mutagenesis of intrinsic genes (3) and the construction of vectors (4) are applications of in vivo recombination in molecular biology. Next to Escherichia coli, S. cerevisiae is the most commonly used host organism in directed evolution (5–9). Its well developed recombination apparatus facilitates mutant library construction. Ligation of mutant genes into expression vectors is in many cases a tedious and non-robust step that needs fine tuning for new plasmid-gene combinations. Yeast gap repair can substitute for ligation to give more reliable high transformation frequency and shortening the protocol for library expression. In gap repair, the mutant gene inserts are cotransformed with open plasmid that contains sequences homologous to the ends of the inserts on both ends. Homologous recombination combines these to form complete plasmids.KeywordsHomologous RecombinationDirected EvolutionBeneficial MutationMutagenic PrimerLithium AcetateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.