We report a simple in vivo technique for introducing an antibiotic resistance marker into phage λ. This technique could be used for direct selection of lysogens harboring recobminant phages from the Kohara λ bank (a collection of ordered λ clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and λ DNA packaging to replace the nonessential λ DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 λ lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara λ phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the λ clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the λ prophage can be readily distinguished from insertion into bacterial chromosomal sequences.
All Science Journal Classification (ASJC) codes
- Molecular Biology