Mapping Genes Controlling Bacterial Cell Permeability Using TraDISort

Bacterial cell envelopes have evolved to control the passage of small molecules into and out of the cell. Understanding these barriers and the pathways that small molecules can take as they cross the barrier could help to improve outcomes in broad areas of applied microbiology, e.g., to improve antibiotic uptake for infectious disease treatment, to control pollutant uptake in bioremediation, and to enhance bioproduct export in industrial fermentation. We developed an innovative high-throughput screening method that can be used to discover novel bacterial genes that affect intracellular accumulation of small molecules, such as efflux or uptake transporter genes, or genes controlling cell membrane composition. This method is a combination of Transposon Directed Insertion site Sequencing and fluorescence-activated cell sorting through flow cytometry (TraDISort). TraDISort has broad applications beyond studies of cell permeability, but in this chapter, we provide a basic protocol to study the penetration of fluorescent compounds.