Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease

Abstract Several non-lethal deletions of the broad host range plasmid RK2 (molecular weight of 7.6 · 10 6 ) have been produced in vitro. The method employed relied on the single Hin dIII restriction nuclease site in RK2 and the ability of phage Mu to insert and thereby add new Hin dIII restriction sites at various positions in the plasmid. The deleted plasmids have in each case lost kanamycin (Km) resistance, and in two cases are defective in self-transmissibility. The method used to reduce the size of the RK2 plasmid also results in the cloning of each of the two ends of the Mu phage DNA on the plasmid derivatives.