Genome-Wide Transposon Mutagenesis in Mycobacterium tuberculosis and Mycobacterium smegmatis
TnSeq, or transposon (Tn) insertion sequencing, is a powerful method for identifying the essential—as well as conditionally essential—regions in a genome, both coding and noncoding. The advent of accessible massively parallel DNA sequencing technologies in particular has resulted in the increased use of TnSeq-based approaches to elucidate various aspects of bacterial physiology and metabolism. Moreover, the availability of detailed protocols has enabled even nonspecialist laboratories to adapt and develop TnSeq approaches to address specific research questions. In this chapter, we describe a recently modified experimental protocol used in our laboratory for TnSeq in the major human pathogen, Mycobacterium tuberculosis, as well as the related non-pathogenic mycobacterium, M. smegmatis. The method, which was developed in close consultation with pioneers in the field of mycobacterial genetics, includes the steps involved in preparing a phage stock, generating a mutant library, selection of the library under a specific experimental condition, isolation of genomic DNA from the pooled population of mutants, amplification of the sites of Tn insertion and, finally, determining the essential genomic regions by next-generation sequencing.
Key wordsTransposon mutagenesis TnSeq analysis MycoMarT7
This work is funded by grants from the South African Medical Research Council (to DFW), the National Research Foundation (to DFW), and the Research Council of Norway through its Centres of Excellence funding scheme, project number 223255 (to MSD). RM is supported by the Howard Hughes Medical Institute through a Senior International Research Scholars grant to Valerie Mizrahi. GM is supported by a grant from the South African Medical Research Council (to DFW) with funds from National Treasury under the Economic Competitiveness and Support Package (MRC-RFA-UFSP-01-2013/CCAMP).
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