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Yeast One Hybrid FAQ

一. Principle of Yeast One-hybrid Experiment

Yeast one-hybrid technology is derived from the yeast two-hybrid GAL4 system, which can analyze and identify whether transcription factors bind to DNA cis-acting elements and verify the interaction sites in yeast cells. In simple terms, a cis-acting element with a known specific sequence is constructed upstream of the minimal promoter (Pmin), and a reporter gene is connected downstream of Pmin to form a bait DNA containing the target DNA Element (bait sequence). The transcription factor is constructed on a vector that can express the AD activation domain to express the fusion prey protein (prey). If the transcription factor and the cis-acting element interact, Pmin will be activated, thereby promoting the expression of the reporter gene. Therefore, whether the cis-acting element and the transcription factor interact can be determined by detecting whether the reporter gene is expressed.

Schematic diagram of yeast one-hybrid-tekbiotech.png

Schematic diagram of yeast single hybrid

二. Common Yeast Single Hybrid Systems

Common yeast single hybrid systems include Y1Hgold (pABAi+pGADT7/pGADT7-REC), Y18 (pHis2+pGADT7/pGADT7-REC), and EGY48 (pLacZi+pB42AD). No obvious advantages or disadvantages were found among the three single hybrid systems. The differences among the three are as follows:

1) pAbAi-Bait (DNA) + pGADT7rec or pGADT7; Y1HGold strain. The pAbAi vector with the target DNA inserted needs to be linearized and transferred to the Y1HGold strain to make the Bait strain; screening marker AbA;

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

3) pLacZi-Bait (DNA) + pB42AD; EGY48 strain. Bait vector needs to be linearized and integrated into the yeast genome; screening marker X-gal.

三. Yeast One-hybrid Experiment Process (taking Y1HGold as an example)

1）Vector:

Yeast single hybrid-tekbiotech1.png

pGADT7 （Connecting transcription factors）

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pGAbAi（Connecting promoter）

2）Host: Y1HGold

3) Culture medium: SD/-Ura, SD/-Ura+different concentrations of AbA, SD/-Ura/-Leu+AbA

4) Experimental steps:

(1) Construct the transcription factor into the pGADT7 vector named AD-prey, and construct the promoter into the vector pGAbAi named AbAi-bait

(2) Linearize the constructed AbAi-bait plasmid with BstbI enzyme digestion so that it can be integrated into the yeast chromosome, and then transfer it into Y1HGold competent cells, spread it onto SD/-Ura nutrient-deficient solid culture medium, and culture it at 28℃ for 2-3 days. Pick the positive clones and perform PCR identification.

(3) Cultivate the positive clones identified in (2) to an OD value of 0.2 and then spread them on SD/-Ura plates containing different concentrations (0-1000 ng/mL) of AbA. Then dilute them tenfold to three concentrations. Using the bacterial solution OD of 0.002 as the standard, screen out the concentration that can inhibit the growth of yeast colonies, which is the AbA concentration that inhibits its self-activation.

(4) Prepare the positive clones obtained in (2) into competent cells

(5) Transfer the empty pGADT7 (control) and the constructed AD-prey (experimental) plasmids into the competent cells prepared in (4) respectively, and spread them on SD/-Ura/-Leu+AbA (AbA concentration is the concentration that inhibits promoter self-activation in (3)) solid culture medium

(6) Observe the growth of the colonies under 28℃ culture conditions. If the control group does not grow but the experimental group grows, it indicates that there is an interaction between the transcription factor and the promoter.

四、Examples of Yeast One-hybrid Applications

Yeast one-hybrid is mainly used in the study of interactions between DNA and proteins (mostly transcription factors):

① Identify whether there is an interaction between DNA and protein

② Screen new genes that bind to cis-acting elements

③ Verify the DNA binding domain of the confirmed DNA binding protein with interaction, and accurately locate the nucleotide sequence that binds to DNA.

Jie Lan et al. used yeast one-hybrid to intercept the W-box of the SLR1 promoter of rice and verified the binding site between the WRKY36 gene and the SLR1 gene.

Yeast single hybrid-tekbiotech3.jpeg

The direct binding of AmMYB24 to MYBCOREATCYCB1 checked by Y1H

In this article AmMYB24 Regulates Floral Terpenoid Biosynthesis Induced by Blue Light in Snapdragon Flowers, the authors used yeast one-hybrid to verify the binding site of the snapdragon MYB24 transcription factor and the OCS promoter.

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