I am doing transcriptional activation activity assay in yeast. To confirm if the two genes I am working on are required for the transcriptional activity, I used pBridge to express my targets. I have confirmed the gene A and B (in pGBK-T7) cannot grow on -T/-H/-A triple DO medium,which indicated A or B alone lacks transcriptional activity. (Meanwhile, I fused GAL4-AD with the BD in pGBK-T7 as a positive control, this construct can survive on -T/-H/-A.) Once I expressed the A and B simultaneously in pBridge, the construct can growth on -T/-H/-M medium. This supported my hypothesis that both genes are required for transcriptional activation activity. The caution is that the AD-BD fusion is able to grow on -T/-H/-M as well. As far as I know, Met is an essential amino acid, at least for human. So my question is if the AD-BD (in pGBK-T7) can survive on medium lacks Met? the strain Y2Hgold is used for the assays.
Y2HGold does not require Met to grow, so yes it can survive on Met- medium. The strain is able to synthesize it.
Hi, Chen. Y187, not Y2Hgold, is a methionine auxotroph. In Y3H system, methionine just control the third protein expression.
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