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284 Tikhmyanova et al.6. Assay of multiple colonies is important, because for some baits, protein expressionlevel is heterogeneous between independent colonies, with accompanyingheterogeneity of apparent ability to activate transcription of the two reporters. Forfurther discussion, see Chapter 16 Fig. 4.7. A replicator for the transfer of multiple colonies can be purchased or easily homemade;it is important that all of the spokes have a flat surface, and that spoke endsare level. A metal frogger can be sterilized by autoclaving or by alcohol/flaming;a plastic replicator must be cut in half to fit to a standard 90-mm Petri plate; it canbe sterilized by autoclaving or rinsing with alcohol. The replicator should have 48spokes in a 6 × 8 configuration. When making prints on a plate, dip the replicatorin the wells of the microtiter plate, then put it on the surface of the solidifiedmedium. Tilt slightly in circular movement, then lift replicator and put it in themicrotiter plate (keep the correct orientation). Make sure all the drops left on thesurface are of approximately the same size. If only one or two drops are missing,it is easy to correct this by dropping approx 3 µL of yeast suspension on the missingspots from the corresponding wells. If many drops are missing, make sure thatall the spokes of the replicator are in good contact with liquid in the microtiterplate (it may be necessary to cut off the side protrusions on the edge spokes of theplastic replicator) and redo the whole plate. Continue replicating by shuttling backand forth between microtiter and media plates. Let the liquid absorb to the agarbefore putting the plates upside down in the incubator. For alternative techniquesto assess LacZ reporters, including growing yeast directly on Xgal- or Xgluccontainingplates, see http://www.fccc.edu/research/labs/golemis/interactiontrapinwork.html.The technique described herein is much more sensitive than a standardXGluc plate assay, and can be done within 24 h of plating on appropriate medium,and is generally preferred in high throughput analysis.8. Transcriptional activation phenotype of bait 1 on the auxotrophic reporters is themost important consideration for library screening, because this is used fordirect selection for interaction phenotype. Therefore, if no activation is detected onLys − plates, one should proceed further; if bait causes considerable growth onLys − plates, it must be modified (e.g., by truncation). There is normally a goodcorrelation between activation of the two reporters, so it is unlikely that a bait,which does not significantly activate LYS2 will significantly activate GusA. Forthe screening strategy described herein, the behavior of the cI bait is mostimportant; as the LexA bait is primarily being used as a counterselection, weakactivation is tolerated.9. In an optimal result, all six colonies assayed for a given transformation would haveessentially the same phenotype. For a small number of baits, this is not the case.The most typical deviation is that of six colonies assayed for a new bait, somefraction appears to be inactive (white in colorimetric assay, and not growing onauxotrophic selection medium), whereas the remaining fraction display somedegree of blueness and growth. Do not automatically select the white, nongrowingcolonies as starting point in a library screen; generally, these colonies possess thephenotypes they do because they are synthesizing little or no bait protein (as can