3.2.1. Library Construction
A variety of cDNA libraries are available commercially or directly from the laboratories where they have been constructed. Although constructing your own cDNA library does offers the opportunity to choose an mRNA source where your protein of interest may have a relevant biological role, cDNA library construction can be both time-consuming and costly.
The authors used the MATCHMAKER library construction kit (Clontech) to generate a cDNA library from Arabidopsis thaliana seedlings. In this protocol, the cDNA library is constructed in one strain of yeast (AH109) by in vivo cloning and the transformants are collected and frozen at -80°C in aliquots. For each yeast two-hybrid screen, the pretransformed cDNA library strain is mixed with the bait strain and diploids are selected (see Note 10). This protocol is advantageous in that only one high-efficiency cDNA library transformation is required for multiple screens with different protein baits. The transformation efficiency is often the bottleneck when performing yeast two-hybrid experiments. A yeast mating protocol leads to more reproducible results and increases the chances of finding rare protein-protein interactions.
A medium-stringency screen by yeast mating is described in which interacting clones are selected by virtue of their ability to grow on medium lacking His and containing necessary levels of 3-AT (see Subheading 3.1.4.) Putative positives are then identified as those that also turn blue in P-galactosidase tests.
1. Inoculate a single, fresh (<2 mo old) Y187 (bait) yeast colony into 10 mL of SD/ -Trp medium and grow overnight at 30°C at 250 rpm in a rotary shaker. The OD600 should be higher than 0.8.
Fig 2. Schematic diagram of the activation domain bait vector pGADT7. The multiple cloning site is shown. (Printed with permission. ©1999 Becton, Dickinson and Company.)
5. Incubate at room temperature for 6 to 8 h (see Note 6). The cells will have settled to the bottom of the tube.
6. Heat-shock the cells for 10 min at 42°C.
7. Plate between 5 and 50 ^L of heat-shocked cells onto selective SD media (see Notes 1 and 2) to select for the presence of the plasmid(s).
8. Incubate the plates for 2 to 4 d at 30°C until colonies appear.
2. Thaw a 1-mL aliquot of your AH109 (library) strain in a water bath at room temperature.
3. In a flat-bottomed 100-mL flask inoculate (a) 9 mL of YPDA, (b) 1 mL of the Y187 (bait) overnight culture, (c) 100 ^L of AH109 (library), and (d) 50 ^g/mL kanamycin.
4. Incubate at 30°C at 40 to 50 rpm in a rotary shaker.
5. After 22 h examine an aliquot using standard light microscopy to confirm the presence of diploids (Fig. 3). If zygotes are present, allow mating to continue for four more hours.
6. To determine the mating efficiency spread 100 ^L of a 1:1000, 1:100, and 1:10 dilution of the mating mixture onto 90 mm plates containing:
7. Store the remaining mating mixture at 4°C.
8. Incubate the mating efficiency plates at 30°C until colonies appear (usually after 2 to 3 d), count the number of colony-forming units (cfu), and calculate the number of viable cfu growing on each type of SD medium:
Vol. plated (^L) x dilution factor no. cfu/mL on SD/-Leu = viability of the AH109 (library) strain no. cfu/mL on SD/-Trp = viability of the Y187 (bait) strain no. cfu/mL on SD/-Leu/-Trp = viability of the diploids (see Note 11)
To calculate the mating efficiency:
no. cfu/mL of diploids x 100 = % diploid no. cfu/mL of limiting partner (see Note 12) Mating efficiencies in our laboratory are typically 40 to 60% (see Note 13).
9. Plate 50 ^L of the mating mixture onto 150 mm SD/-Leu/-Trp/-His/+3-AT plates (approx 30 plates per mating) and incubate at 30°C for 3 to 8 d.
After 2 to 3 d colonies will be visible. Incubate for at least 5 d to allow slower-growing colonies (weak positives) to appear (see Note 14). Choose His+ colonies for further analysis and replica plate onto fresh SD/-Trp/-Leu/-His plates to dilute out extra AD domain plasmids. Grow at 30°C for 2 to 4 d, seal plates, and store at 4°C for up to 1 mo (see Note 15).
3.2.4. Blue (fi-Galactosidase) Testing Yeast
Colonies that grow under His selection (His+) are then tested for fi-galac-tosidase expression. Colonies that are His+ lacZ+ will be characterized as first-round positives (see Note 16).
Here we describe the colony-lift filter assay used in the laboratory to identify first-round positives.
1. Streak out yeast colonies onto selective plates (SD/-Trp/-Leu/-His) and grow for 3 to 4 d. Best results are obtained with fresh growing colonies.
2. Prepare fresh blue test substrate mix. For each plate to be assayed, presoak Whatman no. 5 filter paper in 1.8 mL of blue test substrate mix in a 90-mm plate.
3. Using forceps, place a clean dry filter paper over the surface of the plate of yeast to be assayed. Press the paper onto the cells with a spreader to help the colonies stick to the filter. Use a syringe needle to punch holes through the filter paper and into the agar to orient the filter on the plate.
4. Carefully lift the filter off the agar plate and place into liquid nitrogen face up for 10 s.
5. Allow to thaw and transfer the filter, colony side up, to the plate containing the presoaked substrate filter, being careful to avoid trapping air bubbles.
6. Incubate the filter at 30°C and check periodically for the appearance of blue color (see Note 17).
7. Identify the fi-galactosidase-producing colonies by aligning the filter paper using the orienting marks.
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