Transposon mutants

Leppik R A (1989) Steroid catechol degradation disecoandrostane intermediates accumulated by Pseudomonas transposon mutant strains. J Gen Microbiol 135 1979-1988. 

Watanabe, K. Noda, K., and Maruhashi, K., Enhanced Desulfurization in a Transposon-Mutant Strain of Rhodococcus Erythropolis. Biotechnology Letters, 2003. 25(16) pp. 1299-1304. 

Spontaneous antibiotic-resistant mutants of these organisms are suitable for initial root colonization studies however, transposon mutants will be more suitable for detailed laboratory and field studies. This procedure is useful for sorting out the genetic relationships of these organisms and for determining the mechanisms controlling toxin production. 

Gallagher, L.A. et al. 2007. A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc. Natl. Acad. Sci. USA 104, 1009-1014. 

Tannler, S. et al. (2008) Screening of Bacillus suhtilis transposon mutants with altered riboflavin production. Metab. Eng., 10 (5), 216-226. 

Fig. 2. Strategy for the generation of transposon mutants in Borrelia burgdorferi and identification of the transposon insertion site. Numbers in parenthesis refer to the specific Subheading and step of the Methods section in which the procedure is described.

Confirm that colonies arising under selective pressure are transposon mutants (and not spontaneously-arising antibiotic-resistant variants) by PGR for the presence of the gentamicin-resistance marker. Stab the B. burgdorferi colony with a sterile toothpick, then swirl the toothpick in a tube containing the PGR reaction mix. 

Once confirmed as transposon mutants by PCR, aspirate colonies from the plate using a sterile Pasteur pipet and transfer to liquid BSK medium (5-15 mL) containing appropriate antibiotic(s). Incubate cultures at 35 °C until cells reach high densities (between 5 x 10 and 1 x 10 cells/mL) (see Note 7). 

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