A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology

Abstract

Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is restriction endonuclease- and ligase-independent, and it is fast and easily performed.

Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays

“…In order to improve the efficiency of transferring cDNA fragments into multiple destination vectors, several yeast one-and two-hybrid systems have been modified to use Gateway technology, In-Fusion technology and so on [17][18][19]. Moreover, in recent studies two cDNA libraries were respectively used as bait library and prey library, followed by mating and screening, enabling the screening of multiple libraries in one pool.…”