Low Backgrounds with Aureobasidin A Selection

Clontech’s Matchmaker™ Gold Yeast One-Hybrid Library Screening System provides a simple and efficient method for identifying and characterizing novel protein-DNA interactions (Figure 1). All Matchmaker Gold Systems use Aureobasidin A resistance (AbA^r) as a stringent, highly selective, and easy-to-use reporter. This novel reporter produces very low screening backgrounds since the Aureobasidin A antibiotic (AbA) efficiently kills yeast lacking AbA^r expression.

 

The Matchmaker Gold Yeast One-Hybrid System

In the Matchmaker Gold Yeast One-Hybrid System, tandem repeats of your DNA target/bait sequence, are cloned into the pAbAi reporter vector. To generate your bait-specific reporter strain, the pAbAi vector is then integrated into the genome of Y1HGold yeast using homologous recombination. This strain provides a host for library screening.

 

One-Step Library Construction and Screening

A cDNA library of potential DNA-binding proteins, which are ultimately expressed as fusions to the yeast GAL4 transcription activation domain (GAL4 AD prey proteins), is constructed directly in the Y1HGold[Bait-AbAi] reporter strain using SMART technology and homologous recombination (Figure 2). When a prey protein binds to the bait sequence (Figure 1), its associated GAL4 AD activates AbA^r expression, allowing the cell to grow on medium containing AbA. In library screens, the plasmids encoding the library-derived prey proteins can be easily rescued from the surviving yeast clones and subjected to futher analysis.

 

Get Results Fast!

With Matchmaker Gold, one-hybrid library screening is straightforward, quick and easy:

     Step 1. Create a bait construct by cloning 1−3 tandem repeats of the target DNA-binding sequence into pAbAi.

    
     Step 2. Create a bait-specific reporter strain by the transforming and integrating the linearized pBait-AbAi construct into the Y1H Gold yeast strain and selecting on SD/-Ura agar medium.

    
     Step 3. Confirm the integration of the bait sequence using colony PCR and the Matchmaker Insert Check PCR Mix 1 .

    

   &nbs; Step 4. Use SMART technology to synthesize cDNA containing ends that are homologous to the ends of the linearized pGADT7-Rec vector.

    

     Step 5. Create and screen your library in a single step by cotransforming your bait-specific reporter strain with the SMART-generated cDNA and the linearized pGADT7-Rec vector, and plating on AbA-containing selective medium (Figure 2).

    

     Step 6. Harvest the resulting colonies, which contain putative DNA-binding prey proteins, and analyze your library inserts using the Matchmaker Insert Check PCR Mix 2.

 

Analysis by Colony PCR

Screening positive colonies by PCR is a fast and convenient way to analyze the bait strain and sort through the positive clones identfied in Y1H and Y2H screens. With the new Matchmaker Insert Check PCR Mix 1 you can verify the integration of your Y1H pBait-AbAi construct (Figure 3, Panel A) and with the companion Matchmaker Insert Check PCR Mix 2, you can quickly sort through and analyze the inserts in positive clones that have emerged from either one-hybrid or two-hybrid screens (Figure 3, Panel B).

Yeast One-Hybrid Media Sets

Our one-hybrid Yeast Media Sets each contain a complete set of the all the Yeast Media Pouches you need for Clontech’s Matchmaker™ Gold One-Hybrid protocols, click here for details.

 

 

 

 

Figure 1. Screening for proten-DNA interactions with the Matchmaker Gold One-Hybrid System. One to three copies of the DNA target sequence are cloned into the pAbAi reporter vector, which is then integrated into the Y1HGold genome to create a bait-specific reporter strain. Activation of the AbA resistance gene occurs if a prey protein from the library binds to the bait sequence.

 

 

Figure 2. Use SMART technology and yeast biology to construct and screen your library. Your library is simultaneously constructed and screened directly in yeast. First, SMART cDNA synthesis technology is used to create a pool of cDNA that is flanked by sequence that is homologous to that at the ends of the linearized prey vector, pGADT7-Rec. Next, the newly created Y1HGold-Bait reporter strain is cotransformed with the cDNA pool and pGADT7-Rec, which undergo homologous recombination within the yeast. The yeast cells are then plated n SD/-Leu/+AbA to select for colonies that have an active reporter (i.e., positive Y1H interactions).

 

 

Figure 3. Use colony PCR to quickly analyze your bait-specific reporter strains and sort your positive candidate clones. Panel A: Colony PCR was performed using Matchmaker Insert Check PCR Mix 1 to analyze the insertion sites of Y1HGold pAbAi integrant strains, which contain the pAbAi reporter vector with or without a bait sequence (i.e., three tandem repeats of either the Oct4 or p53 binding site). Lane 1: Y1HGold. Lane 2: Y1HGold [AbAi]. Lane 3: Y1HGold [3xOct4-AbAi]. Lane 4: Y1HGold [3xp53-AbAi]. Lane M: 1 kb ladder. The strain lacking pAbAi produced no PCR product, while those containing pAbAi integrants generated amplicons of approximately 1.4 kb. Panel B: Matchmaker Insert Check PCR Mix 2 was used to amplify prey vector inserts from 15 randomly selected positive coonies obtained using a Matchmaker Gold Screening System (Lanes 3-17). The results allowed the clones to be quickly sorted for further analysis. Lane 1: No template control. Lanes M and M2: 1 kb ladder. Lane M1: 100 bp ladder.