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Dual Bait-Compatible Bacterial Two-Hybrid System 31311. There are options described further in this protocol to introduce the library (asinfectious phage) or the specific library plasmid into the bait-containing reporterstrain. If one of these options is going to be used, then approx 5% of each transformationshould be plated on an LB_C plate to produce “bait only” colonies.After these have grown, they should be transferred to a master plate and preservedwhile other tests are ongoing.12. This is important, because for some baits, protein expression level is heterogeneousbetween independent colonies, with accompanying heterogeneity of apparent abilityto activate transcription of the reporter(s). This is less of a problem with the bacterialtwo-hybrid system than the yeast two-hybrid system, but it is good to be careful.13. When making prints on a plate, dip the replicator in the wells of the microtiterplate, then very gently put it on the surface of the solidified medium. Tilt slightlyin circular movement, then lift replicator and put it back in the microtiter plate(keep the correct orientation). Make sure all the drops left on the surface are ofapproximately the same size. If only one or two drops are missing, this is easy tocorrect by dropping approx 3 µL of yeast suspension on the missing spots fromthe corresponding wells. If many drops are missing, make sure that all the spokesof the replicator are in good contact with liquid in the microtiter plate (it may benecessary to cut off the side protrusions on the edge spokes of the plastic replicator)and redo the whole plate. Continue replicating by shuttling back and forth betweenmicrotiter and media plates. Let the liquid absorb to the agar before putting theplates upside down in the incubator.14. Basal expression levels of some baits (e.g., pBaitC-zip) may be so high that theaddition of IPTG causes no visually discernible difference.15. This initial test includes comparing the candidate clones with the positive andnegative controls characterized in Subheading 3.1.2., step 5; make sure thesecontrols are available fresh at the time of library screening. If the master plate ismore than 1 wk old, restreak and grow anew; if more than 2 wk old, retransform.16. The plating efficiency of positive clones and appearance of background coloniescan be tested by plating dilutions of the positive and negative clones fromSubheading 3.1.2.17. The strongest interactors are not always the most biologically meaningful. InFig. 4B, compare growth of the spots a2, a5, and b5, all of which represent aknown interaction between HRas and RGL-2, with the growth of the spot c3,which represents a previously undescribed (and probably nonphysiological)interaction between HRas and LTBP4.18. The absence of a PCR product from the purified plasmid indicates general PCRproblems (reagents, faulty amplifier). The absence of a PCR product from the positivecontrol E. coli strains (under conditions in which the PCR from the purifiedplasmid works well) indicates a need to adjust the amount of bacteria taken for thereaction (either too low, so there is not enough template, or too high, which inhibitsthe DNA polymerase).19. Perform a restriction digest of up to 10 µL of the unpurified PCR product withHaeIII in a total volume of 20 µL. Rearrange the loading order according to the