ADVANCED BIOREPORTER TECHNOLOGIES FOR TARGETED SENSING OF CHEMICAL AND BIOLOGICAL AGENTS Steven Ripp and Gary S. Sayler
Bioreporters refer to intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment (Figure 1). Bioreporters contain two essential genetic elements, a promoter gene and a reporter gene. The promoter gene is turned on (transcribed) when the target agent is present in the cell’s environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed. In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a Signal reporter gene. Consequently, turning on the promoter gene Transcription Translation Promoter now causes the reporter gene to be turned on. Activation of Reporter mRNA the reporter gene leads to Reporter Gene Protein production of reporter proteins that ultimately generate some type of a detectable signal. Analyte Therefore, the presence of a Figure 1. Anatomy of a bioreporter organism. Upon exposure to a signal indicates that the specific analyte, the promoter/reporter gene complex is transcribed bioreporter has sensed a into messenger RNA (mRNA) and then translated into a reporter particular target agent in its protein that is ultimately responsible for generating a signal. environment. Originally developed for fundamental analysis of factors affecting gene expression, bioreporters were early on applied for the detection of environmental contaminants1 and have since evolved into fields as diverse as medical diagnostics, precision agriculture, food-safety assurance, process monitoring and control, and bio-microelectronic computing. Their versatility stems from the fact that there exist a large number of reporter gene systems that are capable of generating a variety of signals. Additionally, reporter genes can be genetically inserted into bacterial, yeast, plant, and mammalian cells, thereby providing considerable functionality over a wide range of host vectors.
Reporter Gene Systems
Several types of reporter genes are available for use in the construction of bioreporter organisms, and the signals they generate can usually be categorized as either colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical. Although each functions differently, their end product always remains the same – a measurable signal that is proportional to ADVANCED BIOREPORTER TECHNOLOGIES FOR TARGETED SENSING OF CHEMICAL AND BIOLOGICAL AGENTS Page 1 CENTER FOR ENVIRONMENTAL BIOTECHNOLOGY HTTP://WWW.CEB.UTK.EDU
the concentration of the unique chemical or physical agent to which they have been exposed. In some instances, the signal only occurs when a secondary substrate is added to the bioassay (luxAB, Luc, and aequorin). For other bioreporters, the signal must be activated by an external light source (GFP and UMT), and for a select few bioreporters, the signal is completely self-induced, with no exogenous substrate or external activation being required (luxCDABE). The following sections outline in brief some of the reporter gene systems available and their existing applications.
Bacterial luciferase (Lux): Luciferase is a generic name for an enzyme that catalyzes a light-emitting reaction. Luciferases can be found in bacteria, algae, fungi, jellyfish, insects, shrimp, and squid, and the resulting light that these organisms produce is termed bioluminescence. In bacteria, the genes responsible for the lightemitting reaction (the lux genes) have been isolated and used extensively in the construction of bioreporters that emit a blue-green light with a maximum intensity at 490 nm (Figure 2)2. Three variants of lux are available, one that functions at
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