Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

Abstract

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

“…We used the commercially available EZ : : TN transposome system from Epicentre Technologies, which was previously shown to work well for mutagenesis of LVS (Kawula et al, 2004). The transposome consists of a purified transposase enzyme bound to the EZ : : TN transposon, a hyperactive version of the transposon Tn5 characterized by two 19 bp mosaic ends (ME) flanking an antibiotic resistance marker (Goryshin et al, 2000). A complex formed in vitro by the transposase and transposon DNA is stable in the absence of magnesium and can be electroporated into bacteria, where intracellular magnesium ions initiate transposition (Goryshin et al, 2000).…”

Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

“…We used transposition driven by Tn5 transposase derivatives in such a way that we can separate two transposition events, each using different Tnp:transposon end combinations (Naumann and Reznikoff 2002). The first transposition event delivers components for the second transposition event into the chromosome via transposase-DNA complex codelivery (Goryshin et al 2000). The second transposition event generates a deletion or inversion event.…”

“…Electroporation of complexes (Goryshin et al 2000) was done using standard recommended conditions (2.5 Kv, 5mS).…”

Rhodococcus equi

“…A mutagenesis system based on the insertion of a protein-DNA complex of transposase and a DNA fragment harbouring a kanamycin resistance gene flanked by transposase binding sites [28] was shown to integrate efficiently and randomly into the genome of R. equi [48]. Transposome mutagenesis therefore allows the generation of mutant libraries which can be screened for virulence.…”