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  • Title: Site-Specific Mutagenesis of Bacillus subtilis Phage SPO1.
    Author: Stewart CR.
    Journal: Methods Mol Biol; 2019; 1898():57-67. PubMed ID: 30570723.
    Abstract:
    This chapter describes the procedure that we have used to introduce suppressible nonsense mutations into various genes of Bacillus subtilis bacteriophage SPO1. The targeted gene is cloned in a B. subtilis/Escherichia coli shuttle vector. Using an in vitro enzymatic procedure dependent on mutant oligonucleotide primers, a mutation is inserted into the cloned gene, replacing an early lysine codon (AAA or AAG) with a nonsense codon (TAG or TAA). The mutant plasmid is recovered by transformation into E. coli, and is then transformed into B. subtilis carrying a suppressor that inserts lysine at TAG or TAA codons. Recombination is allowed between the mutant plasmid and superinfecting wild-type SPO1, and mutant progeny phage are identified by plaque-lift hybridization to labeled oligonucleotides having the mutant sequence. This procedure is adaptable for other types of mutations, and for other phage-bacteria combinations for which appropriate strains and plasmids are available.
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