The generation of a colE1-Apr cloning vehicle which allows detection of inserted DNA

Abstract

A 3.2 Mdal sequence of DNA, TnA, which contains the ampicillin (Ap) resistance determinant has been translocated from an R plasmid to the plasmid ColE1. A total of 12 isolates were studied. There are at least 8 sites in ColE1 at which TnA has inserted. Insertion at five of these has resulted in a Col^- phenotype. One ColE1-Ap^r plasmid, RSF2124, was examined further and its replication properties are found to be similar to that of the parent plasmid. RSF2124 appears to be a useful plasmid vehicle for the molecular cloning of DNA from diverse prokaryotic sources: it codes for readily detectable Ap resistance and contains a single EcoRI site in a gene affecting colicin biosynthesis so that it is unable to produce colicin upon ligation to other DNA.