4] Applications of gene fusions to green fluorescent protein and flow cytometry to the study of bacterial gene expression in host cells

Abstract

Publisher Summary This chapter describes methodologies that use fluorescent proteins to dissect the molecular basis of microbial infections, including protocols for the use of green florescent protein (GFP) and flow cytometry to identify bacterial genes differentially expressed under varied environments (e.g., host cells and tissues). The chapter also explores the applications of GFP to dissect genetic regulatory networks and methods to quantify bacterial gene expression in host cells. To illustrate these applications two intracellular bacterial pathogens, Salmonella typhimurium and Mycobacterium marinum , are used. GFP is a small fluorescent protein that does not require any exogenous cofactors or substrates for its fluorescence. Therefore, GFP permits the monitoring of gene expression and protein localization in live samples and with single cell resolution. New GFP variants with enhanced solubility and altered emission and excitation spectra are now used routinely for most applications. One such variant, GFPmut , has been described extensively and is the basis of most of the protocols described in the chapter. Bacterial cells expressing GFPmut can be identified easily by examining bacterial colonies under blue light or by looking at bacterial suspensions under a fluorescence microscope with settings and filter sets similar to those used for the routine imaging of fluorescein-stained samples. Any electronic imaging systems capable of detecting fluorescence can be used to screen for GFP-expressing cells. For instance, fluorescent-activated cell sorters (FACS) can rapidly scan cells in suspension, record the fluorescent properties of each cell, and separate them accordingly. Therefore, FACS allows GFP expression to be a “selectable” phenotype.