Creating a Library of Random Transposon Mutants in Leptospira

Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli β2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.