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Dual Bait-Compatible Bacterial Two-Hybrid System 3032. Transform the KJ1567 or Bacteriomatch II E. coli HIS3/aadA reporter strain (and,optionally, the AG58(RP28) LacZ reporter strain) with the following combinationsof plasmids (see Notes 7–11):a. pBaitC-YFG + pLibB-Raf (test for autoactivation).b. pBaitC-Ras + pLibB-Raf (positive control for activation and interaction).c. pBaitC-zip + pLibB-Raf (negative control for activation and interaction).3. Plate each transformation on LB_AC plates and incubate at 37°C for 12–18 h,until colonies have grown.3.1.2. Assessing Bait Activation of Reporter Genes: ReplicaTechnique/GriddingFor each combination of plasmids, assay at least six independent colonies fortheir ability to activate the auxotrophic and colorimetric reporters (see Note 12).Assessment of transcriptional activation requires the transfer of colonies frommaster plates to a variety of selective media. This transfer can be accomplishedsimply, by using a sterile toothpick to move cells from individual patches on themaster plate to each of the selective media. However, in cases in which large numbersof colonies and combinations of bait and prey are to be examined it is usefulto use a transfer technique that facilitates high-throughput analysis. The followingtechnique, based on microtiter plates, is an example of such an approach.1. Add approx 100 µL of sterile NM medium to each well of one half (wells A1–H6)of a 96-well microtiter plate. As shown in Fig. 4A, place an insert grid from a rackof micropipet tips over the top of the microtiter plate, and attach it with tape: theholes in the insert grid should be placed exactly over the wells of the microtiterplate. Although this is not essential, the grid will stabilize the tips in the plate, andallow simultaneous removal, speeding the replication process.2. Using sterile plastic micropipet tips, pick six colonies (1–2-mm diameter) from eachof the transformation plates a–c (Subheading 3.1.1., step 2). Put each set of sixacross one of the first top four rows, and leave the tips supported in a near-verticalposition by the insert grid until all the colonies have been picked.3. Swirl the plate gently to mix the cells into suspension, remove the sealing tape,and lift the insert grid, thereby removing all the tips at once.4. To print the cell suspensions on a plate, place the replicator (see Note 13) into themicrotiter plate, lift it and turn 180°, and then reinsert into the remaining half ofthe plate. This will make an approx 1:20 dilution of one’s primary suspension inthe bottom four rows (reversed mirror image—do not forget).5. Use the replicator to plate bacterial cells suspensions on the following plates seeSubheadings 2.1.–2.3.:a. LB_AC (backup master plate, media test).b. NM_AC (master plate).c. NM_ACI.d. NM_AC_5AT.e. NM_ACI_5AT.