Once constructed, the expression plasmid is transformed into an appropriate yeast strain following standard transformation procedures, as described below. In principle, any laboratory strain with appropriate auxotrophic mutations corresponding to the selectable marker on the expression plasmid can be used as a host strain for expressing Bax. In practice, however, strain background variations may affect the sensitivity of yeast to Bax. We have tested the effect of Bax in a few haploid strains. Some strains, such as BF264-15Dau (70), were found to be relatively more sensitive to Bax, suggesting that strain differences do matter. Thus, it is advisable to test yeast stains from different origins, so that the most sensitive ones can be employed. A second factor to consider when selecting a yeast strain is the availability of auxotrophic markers. URA3, LEU2, HIS3, and TRP1 are the four commonly used auxotrophic markers for S. cerevisiae. Strains with multiple auxotrophic markers provide greater flexibility, especially in cases where the effects of more than one protein are to be tested. Some commonly used laboratory yeast strains contain these auxotrophic mutations, including W303a or a (genotype: MAT a/or a leu2-3 112 his3-ll,15 tryl-1 ura3-l canl-100 ade2-l) and EGY48 (genotype: M A Ta leu2-3 112 his3-ll,15 trpl-1 ura3-l 6LexAop-LEU2). Yeast strains can be obtained from the Yeast Genetic Stock Center at ATCC.
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