Construction of an HpaI and HindII plasmid vector allowing direct selection of transformants harboring recombinant plasmids

Abstract

The construction of the vector plasmid PKN80 is described, which can be used as HpaI or HindII cloning vehicle with direct selection on transformants harboring hybrid plasmids. pKN80 carries the EcoRI.C fragment of phage Mu DNA coding for a killing function which is efficiently expressed upon transformation of pKN80 into Mu-sensitive bacteria. Cloning of DNA fragments at the single HpaI site of pKN80 results in insertional inactivation of the killing function. Whereas religated pKN80 molecules yielded only a few transformants, the transformation efficiency had been increased by a factor of at least ten when HpaI fagments of lambda DNA were added to the linearized vector prior to ligation. More than 90% of the transformants tested containted hybrid plasmids.