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Dual Bait-Compatible Bacterial Two-Hybrid System 305f. NM_ACS.g. NM_ACIS.Incubate the plates at 37°C for up to 12–24 h, then save the NM_AC masterplate at 4°C while assays are run.6. Analysis: ideally, all six colonies assayed representing the same transformationwould have essentially the same phenotype, although some heterogeneity is normal(see Fig. 4B). Bacteria containing the strong positive control (from transformation b)should be detectably growing on HIS3 (NM_AC_5AT) and aadA (NM_ACS)selection plates both with and without IPTG (i.e., even minimal quantities of proteinsproduced should activate these reporters). The negative control (from plasmidcombination c) should not grow on either HIS3 or aadA selection plates. If thebacteria containing the bait under test (from plasmid combination a) shows nogrowth on the HIS3 and aadA selection plates within 24 h, it is probably suitablefor library screening; if it is similar to the positive control, it must be reconfigured.Intermediate growth phenotypes (e.g., weak growth only in plates containingIPTG) suggest the bait may be usable, but may have background in a libraryscreen. Figure 4B shows typical results from a characterization of bacterialreporter activity using the replica technique.Several “technical” conclusions can also be drawn on analysis of the results ofthis experiment. First, it allows optimization of the nonselective minimal medium,which could be especially important for the subsequent library screening.However, LB_AC is a much richer medium than NM_AC; in an optimal result, thedifference in bacterial growth on LB_AC and NM_AC plates should not be dramaticby 12–24 h after plating. If bacterial growth rate and/or plating efficiency on NMis low, it should be optimized (check the components, adjust antibiotic concentrationsand drying time for plates). In the meantime, use the LB_AC plate as a“healthy” master plate. Second, slow growth in the presence of the IPTG-induceron a nonselective plate (NM_ACI) would suggest toxicity of the bait; this maylead to artifacts and many false-positives during a library screen. Third, analysisof the growth pattern of the positive and negative controls allows assessment of theselective medium. If there is no good discrimination between the growth of positiveand negative controls, or if the growth of the positive control cells is very poor at24 h, the concentrations of streptomycin and 3-AT should be adjusted.7. Optional: if the AG58(RP28) LacZ reporter strain is also used, assay β-galactosidaseactivity of the emerging clones by using one of the quantitative assays (e.g., [8,18]).3.1.3. Detection of Bait Protein Expression (Western Blot)Western analysis of lysates of bacteria containing DBD-fused baits is importantfor the characterization of the size and expression level of the bait size. Someproteins may either be synthesized at very low levels, or be posttranslationallyclipped. Either of the above two problems can lead to complications inlibrary screens. Where proteins are proteolytically clipped, screens might inadvertentlybe performed with DBD fused only to the amino-terminal end of thelarger intended bait. Western analysis should be performed as follows: