Construction of Phage Mutants

Abstract

Recent studies have established that the most abundant life form, that of phages, has had major influence on the biosphere, bacterial evolution, bacterial genome, and lateral gene transmission. Importantly the phages have served and continue to serve as valuable model systems. Such studies have led to a renewed interest and activity in the study of phages and their genomes. In order to determine the details of the involvement of phages in these important processes and activities, it is critical to assign specific functions to the phage gene products. The initial functional and gene assignments can be made by general mutagenesis of the phage genomes and of these specific gene products. A very informative mutagenic protocol that has found renewed interest is that using hydroxylamine. This mutagenic protocol has been used to obtain gene mutations involved in the lysogenic cycle of the Salmonella enterica serovar Anatum var. $15+$ phage $\varepsilon^{34}$ (hereafter phage $\varepsilon^{34}$) and to isolate conditional lethal mutants of phage $\varepsilon^{34}$. A similar protocol using plasmid is also described. A plate complementation method is presented to determine quickly the number of genes which are present in the population of mutations isolated from hydroxylamine mutagenesis.