1. What is the experimental principle behind the yeast one-hybrid assay?

Answer: The yeast one-hybrid technique was derived from the yeast two-hybrid GAL4 system and is an effective method for analyzing and identifying the interaction between transcription factors and DNA cis-acting elements within yeast cells. Simply put, a known specific cis-acting element is constructed upstream of the minimal promoter (Pmin), and a reporter gene is linked downstream of the promoter to form a bait DNA containing the target DNA element (bait sequence). The transcription factor is cloned into a vector capable of expressing the AD-activating domain, resulting in the expression of a fusion prey protein. If the cis-acting element binds to the transcription factor, it activates the Pmin promoter, thereby driving reporter gene expression. Therefore, the interaction between the cis-acting element and the transcription factor can be determined by detecting whether the reporter gene is expressed.

2. What are the requirements for bait DNA?

Answer: Bait DNA is typically selected from cis-acting elements or promoter sequences. When using a cis-acting element as bait, it is best to use a short cis-acting element (generally <20 bp), repeated in series 2–3 times to form the bait DNA; if the cis-acting element exceeds 100 bp, a single copy is sufficient. When using a promoter sequence as a bait, it should not contain a TATA box. If the promoter sequence is less than 100 bp, use two copies as the bait. If it exceeds 100 bp, use one copy as the bait.

3. What are the most commonly used basic promoters, and what are their corresponding downstream reporter genes?

Answer: There are three commonly used basic promoters: the PminHIS3 promoter in the pHis2 vector, with HIS3 as the reporter gene; the PCYC1 promoter in the pLacZ vector, with LacZ as the reporter gene; and the iso-1-cytochrome C promoter in the pAbAi vector, with AbAr as the reporter gene.

4. What are the commonly used plasmids and bacterial strains in single-hybrid systems?

Answer: Commonly used plasmids include pHis2, pABAi, pLacZi, pGADT7, pGADT7-REC, and pB42AD. With the exception of pLacZi, all are shuttle plasmids that contain both E. coli and yeast replication origins, as well as various resistance and amino acid deficiency screening markers. pGADT7-REC and pGADT7 have identical functions, but to facilitate library transformation, the former contains a specific sequence that allows for homologous recombination with the library. The pB42AD and pGADT7 vectors are similar in that they both carry the activation domain sequence of the GAL4 transcription factor.

Commonly used strains include Y1HGold, Y187, EGY48, and YM4271. Among these, EGY48 and YM4271 are mating partners and can mate with each other. Select the appropriate yeast strain and corresponding vector based on the experimental protocol.

5. What are the functions of the selection marker and reporter gene, and what are the detection methods?

Answer: The constituent elements of a plasmid include the replication origin, resistance gene, multiclonic site, promoter, primer binding site, and selection marker. In yeast single-hybrid assays, the selection marker in the chosen plasmid is typically an amino acid deficiency marker. Screening using the corresponding deficient medium can determine whether the plasmid has been successfully transformed into the recipient yeast.

In yeast two-hybrid assays, multiple reporter genes are often detected simultaneously, but in single-hybrid assays, there is generally only one reporter gene. By testing whether this reporter gene is expressed under the corresponding screening conditions, one can determine whether Pmin is activated, thereby assessing whether the cis-acting element interacts with the transcription factor. For example, the HIS3 reporter gene is detected using HIS-deficient medium; the LacZ reporter gene is detected using X-gal-color-deficient medium (SD/-Trp/-Ura/Gal/Raf/X-Gal); and the AbAr reporter gene is detected using medium supplemented with AbA.

6. What are the commonly used yeast single-hybrid systems?

Answer: Commonly used yeast single-hybrid systems include Y1Hgold (pABAi+pGADT7 / pGADT7-REC), Y187 (pHis2+pGADT7 / pGADT7-REC), and EGY48 (pLacZi+pB42AD). No significant differences in performance have been observed among these three single-hybrid systems.

7. What are the main differences among the three yeast single-hybrid systems?

Answer:

1) pAbAi-Bait (DNA) + pGADT7rec or pGADT7; Y1HGold strain. The pAbAi vector containing the target DNA must be linearized before being transformed into the Y1HGold strain to create the Bait strain; the AbA marker is used for screening.

2) pHIS2-Bait (DNA) + pGADT7; Y187 strain. The bait does not need to be integrated into the yeast genome and does not need to be linearized; screen for the His+3-AT marker.

3) pLacZi-Bait (DNA) + pB42AD; EGY48 strain. The bait vector must be linearized and integrated into the yeast genome; screen for the X-gal marker.

8. How should the restriction enzyme be selected for linearizing pAbAi-Bait?

Answer: Either BstBl or BbsI can be used to linearize pAbAi-bait; do not use any other restriction enzymes.

9. How can I address the low recovery efficiency of pAbAi-bait restriction digestion products on agarose gels?

Answer: The standard procedure for recovering restriction digestion products is: run on a macroporous gel → cut the gel → lyse the gel → bind → wash → elute. This method yields high-purity digestion products but results in low recovery rates. After digestion, take a small sample for electrophoresis to verify complete digestion (only one band). If digestion is complete, skip the gel electrophoresis → gel cutting → gel lysis steps and proceed directly to binding → washing → elution, which can effectively reduce loss. It is recommended to use at least 1 μg of the vector for digestion.

10. What are the main applications of the yeast one-hybrid assay?

Answer: To verify whether an interaction exists between a known DNA sequence and a protein; to isolate new genes encoding proteins that bind to target cis-regulatory elements or other short DNA-binding sites; to localize the DNA-binding domains of DNA-binding proteins with confirmed interactions; and to precisely identify the nucleotide sequences that bind to DNA.

11. Why is it necessary to test for self-activation in yeast one-hybrid assays?

Answer: The Pmin promoter itself causes background expression of the downstream reporter gene, and the cis-acting elements cloned into the vector may bind to endogenous yeast transcription factors; therefore, self-activation is a common phenomenon. Only when there are no endogenous transcription factors capable of binding to the cis-acting elements—or when their binding ability is very weak—can the background expression of the reporter gene be suppressed, and the single-hybrid assay can proceed.

12. What should be done if the bait exhibits self-activation?

Answer: If pAbAi-bait exhibits self-activation, SD/-Ura plates should be prepared with a concentration gradient of AbA; If pHis2-bait exhibits self-activation, prepare SD/-His/-Trp plates with a concentration gradient of 3-AT (SL0930) to further assess the intensity of self-activation.

13. How should the AbA concentration gradient be set?

Answer: For the initial screening, it is recommended to set AbA concentrations at 0, 50, 100, and 200 ng/mL. Based on the growth of the bait colonies in the initial screening, set the AbA concentration gradient for the next round. For example, if the bait colonies grow in 50 ng/mL medium but not in 100 ng/mL screening medium, you can choose 100 ng/mL as the AbA concentration for subsequent use, or set a gradient between 50 and 100 ng/mL for further screening. Another example: if the bait colony grows in 200 ng/mL medium but grows more weakly than in 0 ng/mL medium, AbA concentrations of 250, 300, 350, and 400 ng/mL can be set for further screening; if, however, the bait colonies show no difference in growth between 200 ng/mL and 0 ng/mL media, further screening can be conducted at 400, 600, and 800 ng/mL. Theoretically, the AbA concentration should not exceed 1,000 ng/mL.

14. How should the bacterial concentration be set when spreading or spotting on plates?

Answer: Whether spreading or spotting, 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, and this should serve as the standard for screening concentrations in repeat experiments. When spreading the culture, the recommended bacterial concentration is OD = 0.002; use 100 μL for a 9-cm petri dish. When plating in spots, 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 three 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.

15. What should be done if self-activation cannot be inhibited?

Answer:

1) First, verify the bacterial seeding method. Experimenters often overlook the impact of bacterial concentration during plate streaking or spotting on autoactivation inhibition results; in fact, bacterial concentration is the primary cause of inconsistent autoactivation inhibition results. Similar to prescribing antibiotics for humans—where the dosage is determined by body weight—the inhibitory effect of the same concentration of AbA or 3-AT varies depending on the number of bacteria.

2) Verify that the auto-activation system is set up correctly. We recommend using pAbAi-P53 as a control, as it is known to exhibit auto-activation and can be inhibited by 100–200 ng/mL of AbA. If inhibition does not occur, this indicates an error in the preparation of the screening medium.

3) If the bait’s self-activation cannot be inhibited even when the AbA concentration reaches 1,000 ng/mL or the 3-AT concentration reaches 80 mM, this indicates that the inserted bait exhibits excessive self-activation, making library screening and validation experiments impossible. The bait must be reconstructed.