Nutrient supplements boost yeast transformation efficiency

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.

Yeast transformation efficiency is enhanced by TORC1‐ and eisosome‐dependent signaling

“…Once the mutants were confirmed by yeast colony PCR, the mutants were cultured on 1× SD “dropout tryptophan” plates for 3 days, followed by growth in the same 1× SD “dropout” medium overnight. Frozen yeast mutant competent cells were then prepared, transformed, and cultured on 1× SD “dropout leucine and tryptophan” plates using the SuccessAA protocol (Yu et al., ). Mutant transformation efficiencies were measured and compared to the efficiency of wild‐type MaV203 yeast cells.…”

“…S. cerevisiae transformations were performed using the SuccessAA protocol (Yu et al., ), an adaptation of the LiAc/SS carrier DNA/PEG method (Gietz, ) with the addition of amino acids in the transformation mix. The concentration of amino acids used was 1.25× the concentration of amino acids found in synthetic complete (SC) medium.…”

“…Frozen yeast mutant competent cells were then prepared, transformed, and cultured on 1× SD "dropout leucine and tryptophan" plates using the SuccessAA protocol (Yu et al, 2016). Mutant transformation efficiencies were measured and compared to the efficiency of wild-type MaV203 yeast cells.…”

“…Recently, we developed a yeast transformation protocol called SuccessAA (Yu et al., ). Using this method, we found that adding nutrients to the transformation and competence reagents substantially increased transformation efficiencies.…”

Yeast transformation efficiency is enhanced by TORC1- and eisosome-dependent signalling

“…Once the mutants were confirmed by yeast colony PCR, the mutants were cultured 488 on 1x YPAD "drop-out Tryptophan" plates for three days, followed by growth in the 489 same 1xYPAD "drop-out" medium overnight. Frozen yeast mutant competent cells 490 were then prepared, transformed, and cultured on 1xYPAD "drop-out leucine and 491 tryptophan" plates using the SuccessAA protocol (Yu, Dawson et al 2016). Mutant 492 transformation efficiencies were measured and compared to the efficiency of wild 493 type MaV203 yeast cells.…”