Two-step cloning of the Escherichia coli regulatory gene ompB, employing phage Mu

Abstract

It is difficult to clone directly some regulatory or structural genes on the basis of their functions, because of their obscure properties or the leakiness of their mutants. To overcome this problem, a two-step cloning method with use of phage Mu was developed and applied to cloning of the ompB gene, an obscure regulatory gene for major outer membrane proteins of Escherichia coli. The ompB gene was first inactivated by phage Mu insertion, and the approximately 25-kilobase (kb) EcoRI fragment, which hybridized with phage Mu DNA, was cloned into a plasmid vector, pBR322. This DNA fragment was considered to contain not only a portion of phage Mu DNA but also a portion of the ompB gene DNA. With this DNA as a probe, the wild-type ompB+ strain was found to contain a 12.7-kb EcoRI fragment which hybridized with the probe. In the second step, this 12.7-kb EcoRI fragment was cloned into a lambda phage vector, lambda 569. Lysogenization of an ompB mutant with this phage suppressed OmpB- phenotypes, indicating that the 12.7-kb EcoRI fragment carried the ompB gene. The same 12.7-kb DNA fragment, as well as a 3.8-kb EcoRI-BamHI subfragment, was cloned into pBR322. Both plasmid clones were able to suppress the OmpB- phenotypes upon transformation of an ompB mutant.