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Site-Specific Mutagenesis of Bacillus subtilis Phage SPO1

  • Charles R. Stewart
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1898)

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.

Key words

Site-specific mutagenesis Bacteriophage SPO1 Bacillus subtilis Primer-directed mutagenesis Recombination Hybridization 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BioSciencesRice UniversityHoustonUSA

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