A visible phagemid system for the estimation of Cre-mediated recombination efficiency.

Abstract

Diversity in phage antibody libraries is important for obtaining useful high-affinity antibodies. The Cre-lox system is generally employed to increase the size of phage antibody libraries. However, estimation of library sizes after Cre-mediated recombination is difficult, since time-consuming nucleotide sequence analyses are required. We have developed a visible phagemid vector system that facilitates the estimation of recombination efficiency between VH and VL genes. In this system, intermolecular recombination between VL genes flanked by incompatible lox sites is coincident with the expression of functional beta-galactosidase. Recombination efficiency can be calculated simply by counting the number of blue colonies on X-gal-containing medium. Molecular analyses of plasmids isolated from blue colonies reveal a novel VH/VL combination. Our results suggest that this newly developed visible phagemid system may be reliably used for the measurement of recombination efficiency, which would enable precise evaluation of the diversity of phage antibody libraries.