Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro.

Abstract

A HindII restriction fragment comprising the Escherichia coli lac regulatory region and the genetic information of the .alpha. peptide of .beta.-galactosidase (.beta.-D-galactoside galactohydrolase, EC 3.2.1.23) was inserted into 1 of the 10 Bsu I cleavage sites of M13 by blunt end ligation. A stable hybrid phage was isolated and identified by its ability to complement the lac .alpha. function. Further characterization of hybrid phage includes retransformation studies, agarose gel electrophoresis, DNA .cntdot. DNA hybridization and heteroduplex mapping. The insertion point was localized at 0.083 map unit on the wild-type circular map, i.e., within the intergenic region. The results prove that part of the intergenic region is nonessential and that the phage can be used as a cloning vehicle.