Reverse Genetic Systems for Leviphages.

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of (i.e., leviphages) infecting . This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-TnGm. This construct was introduced into PAK and HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~10 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.