Generating Libraries of Random lacZY or GFP Gene Fusions in the Bacterial Chromosome: Screening for Differentially Expressed Fusions.

Abstract

Transposable elements engineered to generate random gene fusions in the bacterial chromosome are valuable tools in the study of gene expression. In this protocol, we describe the use of a new series of transposons designed to obtain random fusions to either the lacZY operon or the gene for superfolder green fluorescent protein (sfGFP). Transposition is achieved through the activity of the hyperactive variant of Tn5 transposase (Tnp) whose gene is positioned in cis with respect to the transposable module and under the control of the anyhydrotetracycline (AHTc)-inducible Ptet promoter. The transposable module comprises a promoter-less lacZY operon or the sfGFP gene, with or without the lacZ or sfGFP ribosome-binding site, plus a kan gene for selection. The transposon-transposase unit is harbored on an R6K-based suicide plasmid. The plasmid is introduced into recipient cells by electro-transformation and the synthesis of Tn5 Tnp is induced transiently by including AHTc in the recovery medium. Cells are then plated on kanamycin-supplemented medium (without AHTc) where the plasmid DNA is lost and only cells in which transposition has occurred can form colonies. Fusions are detected by screening for colony color on lactose indicator plates (lacZ transposition) or monitoring green fluorescence (sfGFP transposition). Depending on whether the reporter gene carries or lacks the ribosome binding sequence, the fusions obtained will either be transcriptional or translational. Parallel screening of colonies grown in the absence and presence of a drug (or condition) eliciting a global regulatory response allows identification of fusions specifically activated or repressed as part of such response.