We found that yeast containing the GALBD::NHR-64 fusion protein grew on media lacking histidine, while yeast containing the GALBD construct alone, as well as yeast carrying the truncated NHR-64 lacking the AF-2 domain, were unable to grow on media without histidine (Figure 4D).
Furthermore, the fusion protein corresponding to the ok1957 deletion of NHR-64 was also able to grow on media lacking histidine, although the yeast did not grow as well as those carrying the wild-type NHR-64 fusion protein (Figure 4D).
In order to randomly screen each DBD library we plated an aliquot of the DNA-binding domain library on yeast synthetic media lacking tryptophan at a density that allows the selection of individual yeast colonies.
We selected two-hybrid positives on yeast minimal media lacking tryptophan and leucine (to select for mating events), and lacking histidine and adenine (to select for activation of the two-hybrid reporter genes).
The activation domain strain contains three different Gal4p-responsive reporter genes: GAL2-ADE2 and GAL1-HIS3, which are assayed by selection on yeast synthetic media lacking either adenine or histidine, respectively, and GAL7-lacZ, which can be monitored using colorimetric or luminescent assays for beta-galactosidase activity.
High biomass productivity was also achieved using simplified media lacking expensive inputs such as yeast extract and biotin.
The Arabidopsis gene AtmBAC2, when expressed in yeast, was able to restore growth of the yeast arg11 mutant on media lacking arginine, indicating that AtmBAC2 catalyzes the export of ornithine from mitochondria to the cytosol.
Expression of S. cerevisiae and C. reinhardtii ERG3 cDNA from the ADH1 promoter of yeast expression vector pDD1193 was carried out by culture of the transformed yeast strain in minimal media lacking leucine.
After overnight growth at 30 °C, colonies were replica plated onto synthetic complete media lacking leucine and tryptophan, to select for yeast carrying both plasmids, and incubated at 30 °C.
