Random chromosomal gene disruption in vivo using transposomes

Abstract

Strain engineering of bacteria has been accomplished by many methods where mobile DNA elements (transposons) are inserted into the genomic DNA of a host organism. This chapter addresses engineering with transposable elements complexed with transposase enzyme. In traditional techniques, transposon and transposase are introduced as distinct entities. The method of mobilization into cells is often unique for each class of DNA element, and for each organism. The discovery of pre-formed transposon/transposase complexes (transposomes) that can be electroporated into living cells opens a new gateway to strain mutagenesis. Described are the preparation of electrocompetent bacterial cells and their transformation with transposomes. Once within the cell, the transposome is equipped to randomly insert its DNA into chromosomes without needing additional components. Ocr, a T7 phage protein that inhibits the host restriction of electroporated DNAs, will also be discussed as an adjunct reagent that can widen the applicability of transposomes. The transposomes used in most of the applications are commercially available, but also described is the process of making custom transposon DNAs and transposomes. The techniques are not limited to bacterial strain engineering per se and may be adapted for single-cell eukaryotes as well.