The myth system is based upon the concept of split ubiquitin, which refers to the ability of ubiquitin to be stably separated into end terminal and UBI and C terminal CUB halves capable of reconstitution into a full length pseudo ubiquitin molecule. While this reconstitution is spontaneous when using wild type NUBI introduction of an ISIN 13 to glycine mutation, producing a fragment called NUBG greatly reduces its affinity for CUV, thus blocking pseu ubiquitin formation. If the NUBG and CUB are fused to protein A and protein B respectively and A and B are capable of interaction, then the pseu ubiquitin molecule can once again be formed.
In myth, integral membrane baits are fused to attack consisting of the CUB fragment linked to an artificial transcription factor. While praises are fused to the NUBG fragment, an interaction between the bait and prey leads to the reconstitution of the pseudo ubiquitin, which in turn can be recognized by cytosolic de ubiquitinated enzymes illustrated as scissors. These enzymes cleave after the C terminus of CUB releasing the transcription factor, which can then enter the nucleus and activator reporter system allowing for selective isolation and identification of cells in which BA prey interactions occur.
Hi, I'm Janie Snyder from Igor STAARs Lab in the departments of Molecular genetics and biochemistry at the University of Toronto. Today I'm going to show you a procedure for membrane use two hybrid or myth, and we use this procedure in our lab to study the interactions of integral membrane proteins. So let's get started.
Prior to conducting myth analysis, verify that your bait protein has its N and or C terminus in the cytosol of the cell. The CUB Lex A VP 16 tag must be fused to your protein at such a terminus. Since the de ubiquitinated enzymes necessary for transcription factor release are located in the cytosol, if the topology of your bait protein isn't known, you can generate constructs tagged at both the NNC terminus and using the N-U-B-G-I control test describe shortly, see if either of these is suitable for use in myth, thereby indicating that a particular terminus is cytosolic.
Next, decide which of the two major variants of myth is suitable for your experiment. For native yeast proteins, integrated myth or immy is the method of choice. In Imy Bates are endogenously tagged with the CUB Lex a VP 16 tag, leaving them under the control of their native promoter.
This is advantageous as the wild type expression level of Bates helps eliminate problems associated with protein overexpression, such as an increased number of false positives for non-native yeast proteins, traditional myth or T myth may be used wherein CU B Lex A V VP 16 tag bait are overexpressed ECT topically from a plasmid. We will focus on T myth in this protocol since this form of myth is more widely applicable with the exception of the initial bait construction and the media used, both forms of myth are carried out in an essentially identical manner. The bait must be cloned into an appropriate vector for tagging and expression.
A variety of T myth vectors are currently available such as BV four, p, a, BV four, and PCM BV four, which allow the construction of c terminally tagged Bates, Beit Cub Lexie, VP 16 under the control of a very strong TF one strong A DH one and weak CYC one promoter respectively. Once the team effector has been selected, restriction digests the plasmid at the appropriate restriction site. Cleavage should occur only in the immediate vicinity of the CU B Lxe VP 16 tag upstream of the tag for C terminal tagging or downstream for N terminal tagging.
For example, when using the PAM BV vector, SFI one is an ideal choice, after the plasmid has been digested, stored at minus 20 degrees Celsius until ready for use, the next step is to design primers for the amplification and cloning of the gene of interest. The five prime end of your forward primer must match approximately 35 to 40 nucleotides upstream of the restriction site. While the three prime end must match the first 18 to 20 nucleotides of the target gene, which is a DRB two encoding the beta two and generic receptor.
In our example, the five prime end of the reverse primer must match the reverse complement of approximately 35 to 40 nucleotides downstream of the restriction site. With the three prime end matching the reverse complement of the last 18 to 20 nucleotides of the target gene omitting the stop code on if the CUB Lex say VP 16 tag is being placed at the C terminus as is being done in the example shown, depending upon whether N or C terminal tagging is being performed, ensure that the selected 35 to 40 nucleotides of plasmid sequence used in the forward or reverse primer result in the target gene being cloned in frame with the CU B Lex, a VP 16 tag. Since in our example the C terminus of the A DRB two protein will be tagged.
The 35 basis of A MBV sequence in the reverse primer have been selected such that the A DRB two and CUB gene sequences lie within the same reading frame, amplify the gene of interest by PCR using the selected primers. The PCR parameters will depend upon the particular enzyme and specific primers used to provide an environment in which the gap repair homologous recombination can occur. The previously digested plasmid and the amplified gene of interest are transformed into an appropriate yeast strain using a standard yeast transformation protocol such as that described by geets and woods.
Once the transformed yeast has grown on the plate, pick a single colony of the strain and inoculate it in five milliliters of SD minus leucine. Liquid media grow at 30 degrees Celsius overnight after the culture has grown overnight and reached saturation. Centrifuge the cells at 700 GS per five minutes and remove the supernatant isolate bait plasma DNA from the cell pellet using any commercial mini prep kit.
Follow the standard protocol with one modification in order to ensure sufficient yeast cell lysis at a small volume of 0.5 millimolar soda lime glass beads to the pellet after initial resus suspension and vortex vigorously for five minutes. Then proceed with the commercial protocol as normal. Transform isolated yeast DNA into a chemically competent e coli strain suitable for plasmid propagation with a transformation efficiency of at least one times 10 to the seventh cells per microgram DNA.
After harvesting the plasma DNA from the transformed e coli, verify proper construction of the bait plasmid by sequencing prior to use bait strains must be analyzed to ensure that the bait proteins are properly localized to the yeast membrane. Localization is determined using fluorescence microscopy. Inclusion of A YFP molecule in the bait tag sequence will allow direct visualization of live cells and is commonly used in imy.
Alternatively, a standard immunofluorescence approach using antibody against the Lex A or VP 16 components of the tag can be used after proper localization of the bait has been established. It is necessary to ensure that the bait is not self activating IE, it does not activate the reporter system alone or in the presence of non interacting praise to ensure that the bait is not self activating, we employ the N-U-B-G-I test. In this test.
The bait is transformed with interacting positive and non interacting negative control praise, and then different dilutions of each transformant are spotted on appropriate selective media. Abate must grow on selective media in the presence of the positive control and does not grow in the presence of the negative control in order to be suitable for use in myth. Once the bait has been validated, the myth reporter strain containing the bait can be transformed with a prey library of interest to screen for protein protein interactions.
To begin this large scale yeast transformation inoculate a single colony of the myth reporter strain containing your bait into five milliliters of SD minus leucine media and incubate overnight at 30 degrees Celsius with shaking. Dilute the overnight culture into 200 milliliters of SD minus leucine media and incubate at 30 degrees Celsius with shaking until it reaches an OD 600 of 0.6 to 0.7. When the target OD 600 has been reached, divide the 200 milliliter culture between four 50 milliliter screw cap centrifuge tubes and harvest the cells via centrifugation.
After washing the pellets with sterile doubly distilled water and lithium acetate trissy DTA solution, we suspend each pellet in 600 microliters of lithium acetate trissy DTA solution to each of four 15 milliliter screw centrifuge tubes. Add 2.5 milliliters of PEG lithium acetate solution two 600 microliters of resuspended cells, 100 microliters of salmon sperm DNA solution and seven micrograms of prey library DNA four. Text the tubes for one minute to ensure thorough mixing and then incubate in a 30 degree Celsius water bath for 45 minutes.
Mix briefly every 15 minutes after the 45 minute incubation. Add 160 microliters of dimethyl sulfoxide or DMSO to each tube and mix immediately by inverting the tubes. Incubate in a 42 degree Celsius water bath for 20 minutes.
When the heat shock is complete, collect the cells by centrifugation and resus. Suspend each of the pellets in three milliliters of two X-Y-P-A-D. Pull all the samples together in a single 50 milliliter screw cap centrifuge tube.
Incubate the cells at 30 degrees Celsius for 90 minutes for cell recovery. Centrifuge the cells and resuspend the cell pellet in 4.9 milliliters of sterile 0.9%sodium chloride using 100 microliters of resuspended cells. Prepare tenfold serial dilution in sterile 0.9%sodium chloride ranging from 10 x to 10, 000 x plate, 100 microliters of the 100 x and 1000 x dilution onto selective media and incubate at 30 degrees Celsius for two to three days.
These plates serve as a control and are used to calculate the efficiency of the transformation equally. Divide the remaining 4.8 milliliters of resuspended cells and plate onto large 150 millimeter plates and incubate at 30 degrees Celsius for three to four days. Once colonies have grown, resus suspend single colonies each representing cells containing a potential interacting bait prey pair in 100 microliters of 0.9%sodium chloride and plate five microliter aliquots into selective media containing XG allow to grow for two to four days only.
Colonies that display robust growth and a blue color are selected for further analysis. After isolating and sequencing the plasmids from the positive yeast colonies, compile and analyze all sequencing data to assemble your preliminary list of interactors. To recheck these interactions, the bait dependency test is employed.
In this test, all prey plasms expressing the identified interactors are transformed back into the original bait strain as well as the strain harboring a control artificial bait consisting of a single transmembrane domain fused to the CUB Lex, a VP 16 tag resuspend single colonies from these transformations in 100 microliters of sterile doubly distilled water and spot five microliter volumes under the appropriate selective media plus X sc. Ideally, multiple transformants should be selected for each prey and both the original bait and artificial bait should be spotted onto the same plate. Incubate the plates for two to four days at 30 degrees Celsius yeast carrying the artificial bait in prey that cause activation of the reporter system are considered promiscuous and that specific prey is removed from the list of interactors.
Praise that cause growth and blue coloration in yeast with the bait of interest but not the artificial bait. Confirm a specific interaction. If however, yeast harboring the prey and your bait of interest do not grow.
This prey is removed from the list of interactors. The remaining praise constitute the complete list of interactors identified in the myth screen. Upon completion of the myth procedure, you will have an interact home map.
This represents a collection of candidate interactions that the researcher must further analyze using specific studies determined on a case by case basis. To assess the biological significance of each, We've just shown you how to carry out the membrane E two hybrid or myth procedure to identify interacting partners of a protein of interest. When carrying out the myth procedure, it's important to verify that your be of interest has its end and or C terminus located in the cito of the cell, and to design your tagging strategy accordingly.
Additionally, it's important to carefully assess that your bait strains properly express the bait and that the bait is correctly localized. Additionally, it's important to perform the NUB GI Control test as this is useful for helping to establish screening conditions. Finally, be sure to independently verify all interactions that you detect using myth.
Following these simple steps should ensure that you get the best possible results from the myth procedure. Well, that's it. Thanks for watching and good luck with your experiments.
