Abstract
Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per μg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening.
Highlights
Efficiency of yeast transformation is determined by the rate of yeast endocytosis
The efficiency of yeast transformation in previous research using the LiAc/single-stranded carrier DNA (ss-DNA)/PEG protocol indicated that approximately 1 × 106 colony-forming unit (CFU) per μg plasmid DNA per 108 cells can be expected[4,13]
Previous research indicated that the level of intracellular amino acids can enhance yeast endocytosis via target of rapamycin complex 1 (TORC1) signalling complex[16]
Summary
Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. We found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to CFU per μg plasmid DNA and per cells with a 13.8 kb plasmid DNA This is over 130 times that of current published methods. We investigate some of these factors affecting yeast endocytosis and the effect of adding amino acids and other nutrients (hereafter referred to as nutrient supplements) in the incubation media immediately prior to transformation. This is followed by RSM analysis to optimise the composition of yeast transformation reagent
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