New constructs and strategies for efficient PCR-based gene manipulations in yeast.
Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphylococcus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain.
Puig
O
O
EMBL, Heidelberg, Germany.
Rutz
B
B
Luukkonen
B G
BG
Kandels-Lewis
S
S
Bragado-Nilsson
E
E
Séraphin
B
B
D019747:Aldose-Ketose Isomerases; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A / Q000235:genetics; D014170:Transformation, Genetic
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; D019747:Aldose-Ketose Isomerases; C490353:TRP1 protein, S cerevisiae