View - ResearchGate

Dual Bait-Compatible Bacterial Two-Hybrid System 309with a range of 3-AT concentration allows some ranking of the candidate interactors.1. Pick potential positive colonies directly from the selection plate and resuspend in100 µL of NM medium in a microtiter plate well, using the four upper rows. Also,include positive and negative controls from Subheading 3.1.2.2. Dilute colony suspensions approx 1:30 in approx 100 µL NM, using the four bottomrows of the microtiter plate, as described in Subheading 3.1.2., step 4.3. Using the replica tool, print suspensions and dilutions on the following plates:a. NM_ACI (to confirm effective colony transfer; one master plate).b. NM_ACI_4AT.c. NM_ACI_5AT.d. NM_ACI_10AT.e. NM_ACI_20AT.f. NM_ACIS.g. NM_ACIS_5AT.4. Incubate plates for 24 h at 37°C, and inspect for growth. If necessary, allow platesto incubate an additional 12–48 h at room temperature, inspecting periodically.Compare the growth of the candidate colonies vs positive and negative controls onthe selective plates and rank them accordingly (taking photographs or scans ishelpful). If none of the candidates grow on the streptomycin plates (and particularlyif growth of the positive controls is slow on these plates), replate the wholeset on lower concentrations of streptomycin.5. Colonies that rank best by growth on the 3-AT and/or streptomycin plates should bedesignated as preferred first round positives and carried forward to Subheading 3.3.for further analysis. Keep the NM_ACI master plate in the refrigerator. Glycerolstocks of strongest positives should also be made at this stage (see Note 17).3.3. Second Confirmation of Potential Positive CandidatesIn this stage, candidates initially confirmed as positives in Subheading 3.2.are further tested to determine if the increased reporter-gene expression islinked to the expression of the specific prey isolated from the library. To performthis analysis, the plasmid encoding the prey fusion is isolated and, along withthe bait, is reintroduced into naive selection strain cells. If the ability to grow onselection plates is linked to the prey plasmid, as indicated by recapitulationof the interaction phenotype, then the insert may be sequenced, or used forother tests.3.3.1. Isolation of Purified Prey Plasmid From BacteriaWith Candidate InteractorsA major strength of the optional steps 1 and 2 in this protocol is that it willidentify redundant clones before two rounds of plasmid isolation and bacterialtransformation, which in some cases greatly reduces the amount of work required.