2 Hybrid Screen

Hello All!

I finally performed my yeast two-hyrbid screen a few days ago!

I crossed my bait, the protein E6-AP, with my prey, the human fetal brain cDNA library in it. I had to prepare a concentrated overnight culture of my bait strain. The next day, the library strain was combined with the bait strain. Then the cells were put in a shaking incubator over night. After 24 hours, I plated my mated culture. Plating the mated culture was an ordeal, because I had to pipette 100 μl of the mated culture on each plate. Mind you, I had plate fifty 150 mm plates. These plates were missing certain amino acids, in order to select for true interactions. The yeast will take 5-8 days to grow. In my last blog post, I will let you know how many colonies grew!

The science behind a yeast two-hyrbid screen:

In order to determine a true interaction, the transcription factor needs to bind to an upstream activating sequence, which will in turn, activate the down stream reporter gene. A transcription factor is a protein that can either activate or repress transcription by binding to a specific DNA sequence. In a two-hybrid screen, the transcription factor is divided in two, the bait, which contains the binding domain (BD) and the prey, which contains the activation domain (AD). The binding domain binds to the upstream activating sequence and the activating domain binds to the binding domain, thus activating transcription.

 

I am a visual learner, so I hope that adding pictures can help to explain what I am talking about. In the image above, the last figure, figure D, shows what has to happen on a molecular level, for a true protein-protein interaction to occur. The binding domain binds to the upstream activating sequence and the activating domain binds to the binding domain, thus activating transcription of reporter genes, which enable yeast two hybrid strains to grow on nutritionally selective media. The plates that I plated on also had  X-alpha-Gal on them, which is a chromogenic substrate (meaning it has colored products). The yeast strains that I used produce the enzyme, alpha-galctosidase, when GAL4 transcription is activated. This enzyme leads to the hydrolysis of (breaking chemical bonds by adding water) the X-alpha-Gal in the medium, therefore turning the yeast a blue color. So after my yeast grow on the plates for 5-8 days, I will be looking for blue colonies that were able to grow on the selective media. Each colony that grows should represent a novel protein that interacts with the protein, E6-AP.

Stay tuned for my last post!