Transposon Mutagenesis of M. smegmatis

Mycobacterium smegmatis mc²155 was transformed by electroporation (Parish and Brown, 2008) with the tetR-expressing plasmid pSR125 to produce the strain VTY123 by Hyg^R selection. Next, the MycoTetOP² delivery shuttle plasmid pSR129 was transformed into VTY123 by Gm^R selection. After a 4-hour recovery at 30°C in LB broth, cells were spread on LB plates containing Gm and incubated at 30°C for 4 days. An estimated 88,000 transformants were harvested from the selective medium and suspended at approximately 1.2 × 10⁷ cells per ml (cells/ml) in LB media with Tween 80 by passing through a syringe. This cell suspension was aliquoted and stored at −80°C in 15% glycerol as frozen stocks. To identify transposon mutants, approximately 1.2 × 10⁴ cells from the frozen stocks were spread on an LB agar plate (100 mm × 15 mm) with Kan and ATc. These plates were incubated for 3 days at 39°C, the non-permissive temperature for the ts-ori replication origin (Guilhot et al., 1992; Pelicic et al., 1997). Colonies that formed on these plates were presumed to be transposon mutants without the delivery plasmid.