1: What is the experimental principle behind the yeast two-hybrid assay?

Answer: Simply put, a known gene is cloned into a vector capable of expressing the BD-binding domain, resulting in the expression of a fusion bait protein; the target gene is cloned into a vector capable of expressing the AD-activating domain, resulting in the expression of a fusion prey protein. If the bait and prey proteins interact, the BD-binding domain and the AD-activating domain come into very close spatial proximity, causing the GAL4 transcription factor to undergo reconformation. This restores the transcription factor’s function, allowing it to activate the expression of the reporter gene. By detecting whether or not the reporter gene is expressed, we can infer whether the prey and bait proteins have interacted.

2: What do BD and AD refer to, respectively?

Answer: The GAL4 transcription factor contains two domains: the BD-binding domain at the N-terminus and the AD-activating domain at the C-terminus. The BD-binding domain recognizes and binds to the activation sequence upstream of the target gene, while the AD-activating domain activates the expression of the target gene. In experiments, the bait is abbreviated as BD, and the prey is abbreviated as AD.

3: What is the underlying principle of the GAL4 transcription factor?

Answer: In the GAL4 two-hybrid system, the BD-binding domain and the AD-activation domain are separated. Since the AD-activation domain cannot bind to the activation sequence and the BD-binding domain lacks activation function, the expression of the target gene cannot be activated. By utilizing the interaction and binding of fusion proteins, the AD-activation domain and the BD-binding domain are reconstituted, thereby restoring the function of the GAL4 transcription factor.

4: What are the roles of the selection marker and the reporter gene?

Answer: The components of a plasmid include a replication origin, a resistance gene, a multiclonic site, a promoter, a primer-binding site, and a selection marker. In yeast two-hybrid experiments, the selection marker in the chosen plasmid is typically an amino acid deficiency marker. Screening using the corresponding deficient medium determines whether the plasmid has been successfully transformed into the recipient yeast. Detecting the expression of the reporter gene indicates whether GAL4 transcription has occurred. Reporter gene expression is the result of protein-protein interaction.

5: What are the detection methods for the screening marker and the reporter gene?

Answer: The screening marker is detected through screening on deficient medium. For pGADT7 transformation, the corresponding selection marker is Leu2; for pGBKT7 transformation, the corresponding selection marker is Trp1. When the selection marker is Leu2, a leucine-deficient selection medium (SD/-Leu) is required; when the selection marker is Trp1, a tryptophan-deficient selection medium (SD/-Trp) is required. Detection of the HIS3 and ADE2 reporter genes requires His- and Ade-deficient screening media, respectively. The MEL1 reporter gene requires detection with X-α-gal, the lacZ reporter gene requires detection with X-gal, and the AbAr reporter gene requires detection with aurantiamine (AbA).

6: What are the commonly used plasmids and yeast strains for the GAL4 double-hybrid system?

Answer: The commonly used vectors are pGBKT7 and pGADT7. Both are shuttle vectors that contain replication origins for both E. coli and yeast, as well as different resistance and amino acid deficiency screening markers. Common strains include Y2HGold, AH109, and Y187. Simply select the appropriate yeast strain based on your experimental protocol.

7: How do I choose a strain?

Answer: Saccharomyces cerevisiae mating is a classic example of cell-to-cell fusion. Saccharomyces cerevisiae has two distinct haploid cell types: the MATa (a) type and the MATα (α) type. When cells of these two mating types come into close proximity, they can fuse to form a diploid cell (MATa/α type). Both Y2HGold and AH109 are of the a type, while Y187 is of the α type. Therefore, Y2HGold and AH109 can both fuse with Y187, but AH109 and Y2HGold cannot fuse with each other. If performing yeast two-hybrid using the mating method, you must select the Y187 yeast strain; either Y2HGold or AH109 can be used. If performing yeast two-hybrid using the co-transformation method, either Y2HGold or AH109 can be used; there is no need to select the Y187 yeast strain.

8: What is the difference between AH109 and Y2HGold?

Answer: For two-hybrid experiments, you only need to choose either AH109 or Y2HGold; all experimental procedures are the same. However, the reporter genes differ between Y2HGold and AH109. Their shared screening markers are Trp1 and Leu2, and their shared reporter genes are HIS3, ADE2, and MEL1. The reporter gene specific to AH109 is lacZ, while the reporter gene specific to Y2HGold is AbAr.

9: What do “co-transformation” and “mating” mean?

A: Both “co-transformation” and “mating” refer to experimental methods for integrating the bait and prey into the same yeast cell. Co-transformation involves simultaneously transforming two plasmids (one of which may be a library plasmid) into the same yeast cell; mating involves transforming two plasmids (one of which may be a library plasmid) into two different types of yeast cells (type a and type α), respectively, and utilizing the ability of these two different types of yeast cells to fuse into a diploid cell to construct the two plasmids into a single yeast cell.

10: Under what conditions should the co-transformation method be chosen?

Answer: For interaction validation, the co-transformation method is recommended. Select Y2HGold or AH109 yeast competent cells, transform both the BD and AD plasmids containing the target gene into the competent cells simultaneously, and then screen for transformants using a double-deficient medium (SD/-Leu-Trp). Compared to stepwise transformation and mating, co-transformation can be completed in just 3 days, making it the simplest method. For library screening—whether using a plasmid library or a cDNA library—co-transformation is also possible, but stepwise transformation is more highly recommended. Stepwise transformation involves first transforming the bait into Y2HGold or AH109 yeast competent cells, and then transforming the library into the competent cells that already contain the bait. Stepwise transformation yields a larger number of transformants, meaning more library genes are involved in the interaction screening.

11: Under what conditions should the mating method be chosen?

Answer: In library screening experiments, if the library is in the form of a yeast library, the mating method is recommended. Transfer the bait into Y2HGold or AH109, culture it on SD/-Trp medium, and prepare bait cells at a specific concentration. Then, culture the bait and library cells under specific conditions to fuse them into diploid cells, thereby achieving the goal of simultaneously transforming the bait and prey into the same yeast cell.

12: What are the main applications of the yeast two-hybrid system?

Answer: To discover proteins that interact with known proteins (typically achieved through library screening); to confirm interactions between two proteins; and to identify regions involved in protein-protein interactions.

13: Why is it necessary to test for self-activation in the yeast two-hybrid system?

Answer: Theoretically, BD can bind to the UAS activation sequence upstream of GAL4 on its own, but it cannot trigger transcription. However, if a transcription factor with transcriptional activation activity is cloned into the BD vector, and the bait protein produced by its expression binds to UAS on its own and triggers transcription of the downstream reporter gene, this indicates that the bait exhibits self-activation. Additionally, certain endogenous proteins in yeast cells that possess transcriptional activation activity may also interact with the bait protein. In such cases, it is impossible to determine whether the expression of the reporter gene is due to the interaction between the bait and the prey. Therefore, it is only possible to determine whether the bait and prey interact after ruling out self-activation. In interaction validation, if self-activation testing is not performed and no control group is established—and interaction validation is conducted directly—it is impossible to determine whether the experimental results are reliable; in library screening experiments, if self-activation testing is not performed and library screening is conducted directly, it is highly likely that a large number of false-positive transformants will be obtained, increasing the difficulty of subsequent data processing.

14: How should one select an appropriate reporter gene for autoactivation testing?

Answer: The yeast two-hybrid assay is a complex experiment that can be broken down into several independent sub-experiments, such as autoactivation testing, subsequent library screening, or validation experiments. First, determine the initial screening criteria for the library screening or validation experiment—that is, which reporter gene to detect—and then determine whether the bait exhibits autoactivation by testing whether it can activate that reporter gene. For example, if the autoactivation assay targets the HIS3 reporter gene, but the library screening targets the AbAr and MEL1 reporter genes, then this autoactivation assay cannot be used for library screening.

15: What are the commonly used reporter genes for autoactivation assays?

A: Bait autoactivation is detected through the expression of a reporter gene. Dual-hybrid reporter genes include HIS3, ADE2, MEL1, lacZ, and AbAr; theoretically, any reporter gene can be used for autoactivation detection. However, the only commonly used reporter genes for dual-hybrid autoactivation detection are the AbAr+MEL1 combination or HIS3. With current experimental techniques, it is easier to obtain inhibitors for autoactivation caused by AbAr and HIS3.

16: Why can AbA (aurin) inhibit autoactivation?

Answer: The mechanism of action of AbA is to inhibit the activity of the inositol phosphorylceramide (IPC) synthase encoded by the AUR1 gene in yeast, thereby disrupting sphingolipid synthesis and killing the strain. When the Y2HGold reporter gene AbAr is activated, it confers resistance to AbA in yeast. At the same time, AbA (golden tricholide) can suppress the self-activation of the AbAr reporter gene within a certain range.

17: Why does 3-AT inhibit self-activation?

Answer: 3-AT is a competitive inhibitor of histidine and can suppress the leakage expression of HIS3 and mild self-activation.

18: What are the advantages and limitations of using the AbAr+MEL1 combination for self-activation detection?

Answer: The AbAr+MEL1 combination refers to determining whether a bait exhibits self-activation by detecting whether the reporter genes AbAr and MEL1 are activated. Using SD/-Trp+AbA+x-α-gal for dual-reporter screening allows for a balance between transformation efficiency and false-positive rate.

This combination is only suitable for Y2HGold yeast (not suitable for AH109 yeast).

19: What are the advantages and limitations of using HIS3 for autoactivation testing?

Answer: HIS3 refers to the method of determining whether a bait exhibits autoactivation by detecting whether the HIS3 reporter gene is activated; this reporter gene is suitable for both Y2HGold and AH109 yeast strains. When using Y2HGold yeast, both methods of autoactivation detection are viable; however, when using AH109 yeast, only the HIS3 autoactivation assay can be used. Since the HIS3 screening marker exhibits leakage expression, screening typically requires the use of a certain concentration of 3-AT, making the experimental preparation slightly more complex compared to the AbAr+MEL1 assay.

20: What are some recommendations for designing an AbAr+MEL1 combination self-activation assay?

A: Transform BD-bait into Y2HGold competent cells, then plate them separately onto SD/-Trp and SD/-Trp+AbA+x-α-gal (with an initial AbA concentration of 100 ng/mL) plates for detection. Incubate for about 3 days. If no transformants grow on the SD/-Trp+AbA+x-α-gal plate, or if the number of transformants is significantly lower than on the SD/-Trp plate and the plate does not turn blue, it can be concluded that the bait does not self-activate or that AbA at this concentration inhibits self-activation; If the colony counts on the two plates are similar, and the colonies on the SD/-Trp+AbA+x-α-gal plate turn blue, it is concluded that the bait exhibits self-activation.

Baits without self-activation can be used directly in subsequent experiments. For baits exhibiting self-activation, set up an AbA concentration gradient for screening. If AbA at a certain concentration (<1000 ng/mL) inhibits the growth of the bait strain, select that concentration of AbA as the screening concentration for subsequent experiments.

21: What recommendations are there for designing the HIS3 self-activation assay?

Answer: Transform BD-bait into Y2HGold/AH109 competent cells, then plate them onto SD/-Trp, SD/-His-Trp, and SD/-His+3-AT (with a concentration gradient of 3-AT) plates for testing. After approximately 3 days of incubation, if a reasonable number and size of transformants grow on the SD/-Trp plates but no transformants grow on the SD/-His-Trp plates, it can be concluded that the bait does not exhibit self-activation; If transformants grow on the SD/-His-Trp plate but not on the SD/-His-Trp+3-AT (concentration X) plate, this indicates that the bait is self-activating, but 3-AT at concentration X can inhibit this self-activation. Select this concentration of 3-AT as the screening concentration for subsequent experiments.

22: How should the concentration gradients for AbA and 3-AT be set, and is there an upper limit?

Answer: Although the concentration ranges reported in different sources vary, they are generally similar. The concentration range for AbA is 0–1,000 ng/mL; in principle, use the lowest possible concentration of AbA, with a standard concentration of 200 ng/mL. The concentration range for 3-AT is 0–80 mM, with a standard concentration of 2.5–30 mM. For preliminary experiments, set the initial screening concentration of AbA to 200 ng/mL and the initial screening concentration of 3-AT to 2.5 mM. If self-activation occurs, set up a screening with a wider concentration gradient to identify the optimal concentration. If increasing the AbA concentration to 1,000 ng/mL or the 3-AT concentration to 80 mM fails to inhibit the growth of the bait strain, this indicates that the bait exhibits excessive self-activation, making it unsuitable for yeast hybridassay.

23: For interaction validation experiments, are there any recommended rapid self-activation detection protocols?

A: For interaction validation experiments, it is recommended to conduct self-activation and interaction experiments simultaneously to maximize experimental progress and obtain reliable conclusions. The specific protocol is as follows:

1) Mix the BD-bait and AD-prey plasmids to form the experimental group, and mix the BD-bait with the empty AD plasmid to form the self-activation group. Co-transform the self-activation group and the experimental group into Y2HGold or AH109 competent cells, respectively.

2) Screen for transformants using SD/-Leu-Trp plates, then expand the transformants in SD/-Leu-Trp liquid medium.

3) Dilute the bacterial culture into multiple gradients and plate them onto the same interaction validation deficient medium plate; simultaneously plate SD/-Leu-Trp plates as controls.

4) Analyze the results. If there is no difference in colony growth between the self-activating group and the experimental group on the SD/-Leu-Trp plate, but the self-activating group does not grow while the experimental group grows on the interaction validation plate, this indicates that the bait has no self-activation and the interaction with the experimental group is valid; if both the self-activating group and the experimental group grow on the interaction validation plate, this indicates that the bait has self-activation and it is impossible to determine whether an interaction with the experimental group exists.

5) If self-activation is present, add gradient concentrations of 3-AT or AbA to the interaction validation plates. If, on a particular interaction validation plate, the self-activation group shows no growth while the experimental group grows, this indicates that the bait exhibits self-activation but can be inhibited by that concentration of 3-AT or AbA, and that the interaction in the experimental group is valid. If neither the self-activation group nor the experimental group grows at a specific concentration, this indicates that the bait exhibits self-activation; although it can be inhibited by that concentration of 3-AT or AbA, the experimental group does not interact. If both the self-activation group and the experimental group consistently grow and show similar growth trends, this indicates that the bait’s self-activation is too strong to be used in interaction validation experiments.

24: Why do inconsistent results for auto-activation inhibition occur with the same bait?

Answer: Experimenters often overlook the impact of bacterial concentration during plate streaking or spot seeding on auto-activation inhibition results. In fact, bacterial concentration is the primary cause of inconsistent results. Similar to prescribing antibiotics for humans—where the dosage should be determined based on body weight—the inhibitory effect of the same concentration of AbA or 3-AT varies depending on the number of bacteria.

25: How should the bacterial concentration be determined for plate smears or spot tests?

Answer: For plate smears or spot tests, the resulting colonies should be single colonies. Regardless of how densely they appear, as long as the colonies on the plate are single—neither forming a continuous layer nor clumping together—the bacterial concentration is within a reasonable range. The screening concentration for repeat experiments should be based on this. For spread plating, the recommended bacterial concentration is OD = 0.002; apply 100 μL to a 9-cm petri dish. For spot plating, control the initial bacterial concentration to an OD value between 0.1 and 0.5 (typically set at 0.2), then dilute further in a 10-fold gradient to obtain three additional concentrations, applying 10 μL of each concentration. The effective concentrations of AbA or 3-AT are determined solely based on the screening results obtained at a bacterial concentration of OD = 0.0002; at this concentration, 20 single colonies can theoretically be obtained on a non-selective agar plate.