Phage transposon mutagenesis.

TitlePhage transposon mutagenesis.
Publication TypeJournal Article
Year of Publication2009
AuthorsM Siegrist S, Rubin EJ
JournalMethods Mol Biol
Date Published2009
KeywordsBacteriophages, DNA Transposable Elements, Mutagenesis, Mutation, Mycobacterium, Transduction, Genetic

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Alternate JournalMethods Mol. Biol.
PubMed ID20560067