This chapter describes the methods and application of the Tn3 shuttle mutagenesis system. Tn3-free vectors can be used for random insertions scattered throughout the genome and used to screen for specific phenotypes linked to the insertion mutations. In shuttle mutagenesis procedure, gene (YFG) is cloned into a Tn3-free vector. A vector containing YFG (pHSS-YFG) is transformed into a pLBl01-containing strain. The resulting strain contains the pHSS-YFG target molecule and the pLB101 transposase-produeing plasmid. The transformant is then mated with a strain containing the conjugal plasmid pOX38 into which a defective minitransposon has been previously placed (pOX38::m-Tn3). Plasmid pOX38 acts as adelivery vehicle for the transposon. Transconjugants contain cointegrates between pOX38::mini-Tn3 and pHSS-YFG. The cointegmte is then resolved by conjugation with an F- strain that expresses the Cre protein from phage P1. The Cre enzyme is able to catalyze a site-specific recombination/resolution event at loxP sites carried on the Tn3-derived minitransposons. The products formed from cointegrate resolution are the original pOX38::mini-Tn3 and the target plasmid with a minitransposon insertion.
ASJC Scopus subject areas
- Molecular Biology