Uma tela de 2-híbrido de levedura em lote para comparar as interações da proteína

This method uses the principle of the yeast 2-hybrid system in both populations to identify protein-protein interactions that answer key questions about a variety of molecular and cellular processes. This technique leverages deep sequencing to see the interactions of one protein versus another. Since this approach is geared to make parallel comparisons, it can identify sets of proteins that interact with one protein versus another or different confirmations of the same protein or while typing a disease causing mutant version, to reveal differential interactomes.

A new yeast 2-hybrid bait plasmid pTEF-GBD was constructed for this procedure as described in the text protocol. This plasmid produces Gal4 DNA binding domain fusion proteins within a TRP1 centromere based low copy plasmid carrying the kanamycin resistance gene that also allows cloning of bait fragments both upstream and downstream of the Gal4 DNA binding domain. This bait plasmid was used to transform MATa yeast, and expression of the Gal4 bait fusion proteins was verified by immunoblotting with anti-MIC antibodies.

In addition, a new yeast 2-hybrid library in the streamlined prey plasmid vector PGAL4 AD was created. Genomic DNA was fragmented by shearing, size selected, modified with adapters, and inserted into PGAL4-AD to create the saxairtab yeast 2-hybrid library. To begin the procedure for creating yeast populations with bait and prey libraries, inoculate three milliliter cultures of each of the MATa yeast transformants carrying the various trip one containing bait plasmids and CSM minus trip medium.

Include two separate cultures, containing the vector plasmid alone, to serve as procedural controls. Incubate the cultures at 30 degrees Celsius and 200 rpm for six hours. Then dilute each culture into a 25 milliliter culture in a sterile Erlenmeyer flask and incubate overnight.

Thaw a frozen vile of MAT alpha cells containing the lu2-carrying prey library at room temperature. Use the entire thawed vile of cells to inoculate 125 milliliters of CSM minus lu minus met medium in a sterile Erlenmeyer flask. Grow the culture at 30 degrees Celsius and 200 rpm overnight.

On the following day, after confirming that the OD600 of the overnight mat a transformant cultures range between 1.0 to 1.5, transfer the cultures to 50 milliliter conical tubes and centrifuge 21 OD 600 equivalents of each culture at 4, 696 times g at room temperature for five minutes. Resuspend each pellet of mat a cells in 10 milliliters of sterile water, transfer the suspension to a new 50 milliliter conical tube, and centrifuge at 4, 696 times g at room temperature for five minutes. Using a pipette, gently remove the supernatant, without disrupting the pelletted cells, and resuspend the pellet in four milliliters of buffered YPDA medium at pH 3.7.

For every 10 mating reactions desired, pellet 39 OD 600 equivalents of the mat alpha strain carrying the library plasmids in a 50 milliliter conical tube. Resuspend the mat alpha cells in 10 milliliters of sterile water and repellet in a new 50 milliliter conical tube at 4, 696 times g at room temperature for five minutes. Using a pipette, gently remove the supernatant, without disrupting the pelletted cells, and resuspend the pellet in 10 milliliters of buffered YPDA medium at pH 3.7.

To set up each mating reaction, add the following to a new 50 milliliter conical tube:One milliliter of MAT-A transformed cells, one milliliter of MAT-alpha library containing cells, and one milliliter of buffered YPDA at pH 3.7. Incubate at 30 degrees Celsius with gentle, orbital agitation for 90 minutes. After 90 minutes, centrifuge the cells at 4, 696 times g at room temperature for five minutes.

Remove the supernatant and resuspend the pellet in two milliliters of one to one buffered YPDA-YPD. Plate all two milliliters of cells onto a 100 milliliter YPD plate and incubate at 30 degrees Celsius for approximately 20 hours. To harvest the cells, pipette two to three milliliters of CSM minus lu minus trip medium onto each YPD plate and use a cell scraper to dislodge the cells.

Pipette the dislodged cells into a 50 milliliter conical tube. Rinse the plate four to five times with two to three milliliters of CSM minus lu minus trip medium by using a 1, 000 microliter pipette to pipette up the medium and gently ejecting it across the YPD plate surface. Centrifuge the cells at 4, 696 times g at room temperature for five minutes.

Discard the supernatant from each conical tube and resuspend the cells in 40 milliliters of CSM minus lu minus trip medium by pipetting up and down. To estimate the number of diploid cells formed, dilute four microliters of the diploid mixture into 200 microliters and 2, 000 microliters of CSM minus lu minus trip medium, respectively. Plate 200 microliters of each dilution onto a CSM minus lu minus trip plate to represent a one to 10, 000 and one to 100, 000 full dilution of the stock of diploids harvested.

Incubate the plates at 30 degrees Celsius for 36 to 40 hours. Immediately use the remainder of each 40 milliliters of cell resuspension to inoculate 500 milliliters of CSM minus lu minus trip medium in a one liter Erlenmeyer flask. Measure the OD 600 of each culture.

Incubate these flasks at 30 degrees Celsius, with shaking at 180 rpm, until they reach saturation, which typically takes 36 to 40 hours. Monitor cell growth at 24 hours and 36 hours by measuring the OD 600. Before continuing with the procedure, confirm the mating efficiency of the two yeast strains.

A minimum number of 200 colonies on the one to 10, 000 dilution plate is required. Once the cultures have reached saturation, use a pipette to remove a 20 milliliter alloquade from each culture and inoculate two liter Erlenmeyer flasks. One flask containing 750 milliliters of CSM minus lu minus trip medium and a second flask containing 750 milliliters of CSM minus lu minus trip minus his medium.

With the lowest level of three amino 124 triasol that eliminates background, determined previously as described in the text protocol. Mix the new cultures well by swirling. After measuring the initial OD 600, incubate the cultures at 30 degrees Celsius, while shaking at 180 rpm, until saturation, which typically occurs within 24 hours for the unselected CSM minus lu minus trip culture, but can take over 70 hours for cultures under selection for yeast 2-hybrid interactions.

Once cultures have reached saturation, remove 11 milliliters of each culture by pipette and sediment the cells with a five minute spin at 4, 696 times g at room temperature. Discard the supernatant and proceed with DNA extraction as demonstrated in the next section. To begin the DNA extraction procedure, use a pipette to resuspend each cell pellet prepared earlier in 500 microliters of strong TE buffer.

Transfer to a 1.5 milliliter micro-centrifuge tube and add 10 microliters of zimolay stock to each tube. In a fume hood, add three microliters of beta marketol ethanol to each tube and mix well. Incubate in the 37 degree Celsius incubator for 24 to 36 hours.

Next, extract the samples two times with 500 microliters of fenol chloroform isolaymal alcohol as described in the text protocol. Add seven microliters of four molar sodium chloride and 900 microliters of ice cold, 100%ethanol and mix by inversion. Sediment the DNA by spinning in a micro-centrifuge at 21, 130 times g for 10 minutes at room temperature.

Discard the supernatant by pipetting and wash each pellet three times with 900 microliters of 70%ethanol. Sediment the DNA by centrifuging at 21, 130 times g for two minutes. After removing the residual ethanol, dry the pellets at 42 degrees Celsius for seven minutes.

Resuspend each pellet in 120 microliters of 0.1x strong TE in a 37 degree Celsius water bath for 90 minutes. Pipette 60 microliters of the extracted DNA into a sterile 1.5 milliliter micro-centrifuge tube. Add 120 microliters of strong TE and 3.5 microliters of RN ace A stock, flick to mix, and incubate at 37 degrees Celsius for one hour.

Add seven microliters of five molar ammonium acitate and 900 microliters of ice cold, 100%ethanol to each tube. Mix by inversion and sediment the DNA, as demonstrated previously. Resuspend the RNace-A treated DNA in 55 microliters of 0.1x strong TE in a 37 degree Celsius water bath for 90 minutes.

Quantify the DNA by absorbance at 260 nanometers. The next step is to perform PCR on the CDNA inserts. Perform two 50 microliter PCR reactions per DNA sample.

To each reaction add water, five micrograms of DNA sample, 25 picamoles of each forward and reverse primer matching the prey library plasmid, and 25 microliters of high fidelity 2x PCR master mix. Amplify the reactions with this program. A 30 second de-naturation at 98 degrees Celsius, 25 cycles of D-natrine at 98 degrees Celsius for 10 seconds, anealing at 55 degrees Celsius for 30 seconds, and extension times of three minutes at 72 degrees Celsius, followed by a five minute incubation at 72 degrees Celsius.

Analyze each PCR reaction by 1%DNA agrose chill electrophoresis with a gel containing athidiumbromine and visualize the gel by UV transillumination. Samples without selection show a generalized smear of DNA, but upon selection individual bands can be discerned. Combine duplicate PCR samples and purify using a kit following the manufacturers instructions.

Quantify the DNA by absorbance at 260 nanometers on a spectrophotometer. Subsequently, perform deep sequencing, followed by bioinformatics processing and verification as described in the text protocol. The new random fragmentised yeast 2-hybrid library was used in the demonstrated procedure and the plasmid inserts amplified by PCR were analyzed by DEEP sequencing and bioinformatics processing using the DEEPN software and compared with a previously published library.

This plot shows the rank order of reeds per each gene in the libraries divided by reeds per gene found by sequencing the yeast genome. This histogram shows the number of unique plasmids per gene that encodes a fragment that is both in the protein coding region and in the proper translational reading frame. A comparison of the numbers of different plasmids in each library that encode yeast genes and the proportion that are and are not in the correct translational reading frame or are inserted backwards is shown.

Overall, the new library has many more different plasmids. These methods ensure that the library can be reproducibly transferred to cells containing different bait proteins and that the selection procedure can yield reproducible sets of genes that are enriched, distinguishing them as specific candidate-interacting proteins. Once mastered, this technique can be done in about a week if performed properly.

Most of this work is incubating cultures of cells and the actually hands-on time is just a few hours. While attempting this procedure, it's important to remember to create populations of a large number of individual diploid cells so that the library can be reproducibly transferred to cells containing the different bait proteins. It is also important to pay close attention to the dilution conditions and growth rate of cells for those populations undergoing selection for positive yeast 2-hybrid conditions.

Following this procedure, computational methods that are explained in the accompanied work will identify the candidate interacting proteins and provide a path for validating these interactions using a traditional yeast 2-hybrid assay or other biochemical methods. After watching this video, you should have a very good idea about how to reproducibly create diploid yeast populations that contain the prey plasmid library and grow them in batch to select for positive yeast 2-hybrid interactions. Don't forget that working with organic solvents, during the DNA purification protocol, can be dangerous and should be done in the fume hood wearing gloves and a lab coat.