Development of M13 as a Single-stranded Cloning Vector: Insertion of the Tn3 Transposon into the Genome of M13

Abstract

As a first step towards developing phage M13 as a cloning vector we have investigated the in vivo insertion of a transposable ampicillin-resistance element (Hedges and Jacob 1974; Heffron et al. 1975a,b, 1977) into its genome. Passage of M13 through Escherichia coli RSF2124, a strain carrying the element Tn3 inserted into the plasmid ColE1 (So et al. 1975), yielded phage preparations capable of transducing sensitive E. coli strains to ampicillin resistance. By repeated colony purification of transduced cells on ampicillin plates we have isolated clones that produce homogeneous transducing phage capable of transducing ampicillin resistance with high efficiency. We describe here the properties of one of these isolates, which we have termed M13 Tn3–15.

To establish that the transducing activity is associated with the M13 phage produced by resistant cells, we have investigated the ability of antibodies against the M13 coat protein to inactivate both the plaque-forming ability and the transducing activity of the M13 Tn3-15 phage. Table 1 shows that both plaque formation and transduction to drug resistance are inactivated by purified antibodies to the viral coat protein. Gamma globulins from rabbits that had not been injected with coat protein had no effect on either plaque formation or transduction.

Electron microscopic observation of the M13 Tn3-15 phages shows them to have a mean length approximately 1.7 times that of the M13 unit length. Figure 1 shows M13 and M13 Tn3-15 in a mixture of the two phages. Replicative forms (RF) of the M13 Tn3-15 DNA have been isolated...