CHARACTERIZATION OF IN-VIVO REPORTER SYSTEMS FOR GENE-EXPRESSION AND BIOSENSOR APPLICATIONS BASED ON LUXAB LUCIFERASE GENES

Advances in genetic engineering methods have allowed the development o f an increasing number of practical and scientific applications for bi oluminescence with lux genes cloned from a variety of organisms. Biolu minescence derived from the shortened hex operon (luxAB genes) is a co mplex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this rep ort, we describe a two-phase kinetic behavior of the light emission wh ich must be properly taken into account in any quantitative measuremen ts of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and inter preted in terms of the biochemistry underlying the bacterial luciferas e mechanism. We show that the intensity profile of each of the two pha ses of the luminescence signal is responsive (and exhibits different s ensitivities) to the concentration of added decanal and other componen ts of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol d esign, and specific recommendations for using the luxAB system as a mo lecular reporter are presented, along with versatile assay protocols t hat yield meaningful and reproducible signals.