Yeast Two-Hybrid Essay -- Biology, DNA

The initial transformation task required two yeast strains (both as Mav203; Leu–, Trp–, His–), with one containing the pDBLeuDa4 bait plasmid, and the other with just the pDBLeu plasmid. The two strains were each transformed with a pPC86DMID1(Y) prey plasmid, where (Y) referred to a full-length positive control (MID1) , a DBB strain (MID1DBB), and a DCC strain (MID1DCC). The transformation process with the two strains and three prey plasmid DNAs resulted in 6 (2x3) transformation mixtures (Table 1). The transformation mixtures were then spread-plated at 1x and 1/10x dilutions using half of each plate on an SC (synthetic complete) yeast growth medium minus selected amino acid(s); one plate without Leucine (SC-Leu), and two plates without Leucine and Tryptophan (SC-Leu-Trp). Plates were then incubated for 4 days at 30 ° C, however due to failed growth in the transformation plates for pPC86DMID1 (both yeast strains), and an additional set of results had to be obtained. The spread-plated results for each strain, transformation, result source, and their associated control cases can be seen in Table 2, with a â€Å"Yes† growth observation referring to confluent growth and â€Å"No† for non-confluent growth. Following the incubation period, each of the 6 transformation plates were sampled and underwent 4x 10-fold serial dilutions, which were then spot-tested to either an SC-Leu-Trp, or SC-Leu-Trp-His (minus Histidine) medium. Each strain-transformation occupied half of each plate, with a spot per dilution level (Figure 1). An additional set of spot-tests were also performed to test for auto-activation of the a4 bait protein, using a strain transformed with an unmodified pPC86 plasmid (MaV203+pDBLeuDa4+pPC86). Following another round of in... ...the DCC variant (for all spot dilution levels). The lack of growth for the DBB variant suggested the B-box region was a requirement for interaction/binding with the a4 protein, while the coiled-coil domain didn’t appear to be a requirement. Its removal however did result in noticeably decreased growth rates when compared to the full-length control test, suggesting its removed resulted in some level of decreased interaction with a4, whether it usually stabilised the binding process, or is removal disrupted the overall form and binding site is unknown, and would require additional testing. However given the external result source, it could have been that group’s sampling and dilution methods had resulted in decreased cell viability, though a simple retest could rule this out, and would be a good starting point to determine any interactions with the coiled-coil domain.