Norwegian Arabidopsis Research Center

Please contact:
Reidunn B. Aalen
or
Ellen Andersen Department of Molecular Biosciences
Pb 1041, N-0316 Oslo, Norway.

The yeast two-hybrid system is a sensitive in vivo assay for the detection of specific protein-protein interactions. The system allows identification of genes encoding proteins that interact with a target protein. It is based on the modular nature of some eukaryotic transcriptional activators which are characterized by two separable functional domains. One domain interacts with the DNA and must either be covalently linked to, or interact with, a second domain (the activation domain) to cause transcription. The two hybrid system utilizes the yeast GAL4 transcriptional activator which is required for expression of genes encoding proteins involved in galactose metabolism.

• testing interactions between known proteins
• screening libraries for proteins which interact with a known protein
• defining domains and/or amino acids required for an interaction

• We use Clontechs Matchmaker Library Construction & Screening Kit
• Cloning vectors - pGBKT7 and pGBT9 for cloning of bait sequence
• Yeast transformation - of bait, using yeastmaker technology, verifying presence of construct (yeast miniprep, PCR)
• Library construction - available now: library made from flowers and buds
• Screening of library - greater than or equal to 1x10⁷ independent clones are screened for their ability to interact with the bait
• Analysis of positive clones - yeast plasmid miniprep, PCR and restriction analysis of plasmids, frozen stocks
• Sequencing of positive clones
• Retesting of positive interaction

Retesting of positive colonies on selective medium.
Image by Tage Thorstensen