Tips for Screening with Yeast Two Hybrid Systems

Posted by Jessica Welch on Oct 22, 2015 10:30:00 AM

Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions.

The 1980s saw a flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period, proteins were successfully expressed as fusions that retain their individual activities (1). Researchers also discovered the modular format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation (TA) domains retain their individual activities when separated (2), and that DBD and TAs from different systems could be combined effectively (3).

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Topics: Plasmid Technology, Yeast

Tag Your Favorite Yeast Genes with Ease

Posted by Julian Taylor-Parker on Nov 19, 2013 9:37:00 AM

Homologous recombination is the process by which nearly all domains of life repair genomic damage, specifically double strand breaks. Researchers have long taken advantage of this natural process to integrate protein tags into the genomes of S. cerevisiae and S. pombe. The protocol is surprisingly simple, requiring only a PCR product containing the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal site of insertion. The linear PCR product is introduced into the cell by direct transformation. A given insert will typically contain both a protein modification sequence and a selectable gene product for isolation of successful transformants.

Addgene distributes several ready-to-use, modular plasmids, combining fluorescent tags, epitope tags, protease sites, and selection markers. These are especially useful in protein complex studies where tagging of multiple protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated. Simply design your amplification primers with the desired targeting homology—in frame, of course—and start tagging!

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Topics: Plasmid Technology, Hot Plasmids, Genome Engineering, Yeast

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