View - ResearchGate

Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 261Fig. 2. Plasmid maps. (A) pGLS23, used for bait 1 expression. (B) pMW103, forbait 2 expression. (C) pJG4-5, used for library or defined interactor (prey) expression.(D) pLacGus (also known as pDR8), double reporter (cI-responsive GusA and LexAresponsiveLacZ).2. pMW103—plasmid for making LexA-fusion protein (bait 2) (Fig. 2B). Expressionis from the constitutive ADH1 promoter. The yeast selection marker is HIS3 andthe bacterial selective marker is Km R .3. pJG4-5—the plasmid for making a nuclear localization sequence—AD—hemaglutininepitope tag fusion to a unique protein or a cDNA library (21), Fig. 2C. AD-fusionexpression is from the galactokinase (GAL1) galactose-inducible promoter. Theyeast selection marker is TRP1, and the bacterial selective marker is Ap R .4. pLacGus—the reporter plasmids containing 8 LexA operators upstream of theLacZ reporter gene, and 3 cI operators upstream of GusA reporter gene (Fig. 2D).The yeast selection marker is URA3, and the bacterial selective marker is Km R .