Tn7-mediated mutagenesis of Saccharomyces cerevisiae genomic DNA in vitro

This chapter describes in vitro insertional mutagenesis of the complete genome of Saccharomyces cerevisiae by the bacterial transposon Tn7. The in vitro transposition reaction of Tn7 is accomplished with purified components, allowing the mutagenesis of any purified DNA fragment. The chapter reviews the application of this in vitro transposition technique using total yeast genomic DNA to create a library of insertional mutants, which are incorporated into the genome by transformation and homologous recombination. After phenotypic analysis of the resulting mutant collection, the mutagenic insertion site can be determined rapidly and easily. The randomness, coverage, and efficiency of this method are also discussed. Tn7 is a bacterial DNA transposon that transposes via a cut-and-paste mechanism. The Tn7 reaction is mediated by the transposon-encoded proteins, TnsA, TnsB, TnsC, TnsD, and TnsE. TnsABC form the core recombination machinery, and TnsD and E are alternative target site selecting proteins. If insertion is mediated by TnsABC+D in vivo, transposition occurs into the single attTn7 site in the E. coli genome.