Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid

Abstract

A purified fragment of DNA that determines resistance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F''lac. This F''lac fragment, exhibiting a MW of 6 .times. 106, carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-F''km plasmid [designated pML31] also retains the incompatibility properties of the parent F''lac plasmid. Large amounts of the kanamycin resistance fragment are obtained as a selective agent for this procedure from a hybrid plasmid that was constructed by combining the EcoRI-generated kanamycin resistance fragment of a MW of 4.5 .times. 106 with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid E1 that has a MW of 2.2 .times. 106. The recombinant plasmid replicates extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA are obtained. The substantial difference in size between the 2 fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis.