Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-10-06
1997-11-04
Guzo, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 8, 435 29, 43525233, 536 232, 536 237, 536 241, C12Q 168, C12N 121, C07H 2104
Patent
active
056838689
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The invention relates to the detection of environmental insults at levels below those necessary to compromise cell metabolism. More specifically the invention provides a transformed bacterial host containing a DNA construct comprising a stress inducible promoter operably connected to a reporter gene or gene complex such that the presence of an environmental insult will induce the expression of the reporter genes. The preferred reporter genes are those that are responsible for bacterial bioluminescence.
BACKGROUND
Increasing public concern and mounting government regulations have provided impetus for the development of environmental sensing systems capable of detecting contaminants in soil and ground water. Highly sensitive and specific detection systems incorporating analytical tools such as Gas Chromatography and Mass spectrophotometry have been known for several years; however, these systems require expensive equipment and skilled operators. Moreover, sample preparation and data analysis is often cumbersome and time consuming. Toxicity assays involving living organisms such as Daphnia, used in the standard U.S. water toxicity test, are simpler; however, these tests are non-specific and not particularly rapid. Somewhat more rapid are cell based toxicity assays that incorporate a bacterial cell as the sensitive element. These systems use bacterial cells as a reagent in a conventional automated analytical system. For example the RODTOX system (Central Kagaku., Tokyo, Japan) is a batch assay that measures bacterial oxygen consumption and was designed for use in sewage plants. Other bacteria based systems such as the GBI TOXALARM system (Genossenschaft Berliner Ingenieuirkollective, Berlin, Germany) can measure the presence of specific chemicals. The GBI TOXALARM is known to be able to detect the presence of as little as 0.1 ppm potassium cyanide in a sample. These detection systems are useful, but are hampered by cumbersome and complex detection systems. Recently, the phenomenon of bacterial bioluminescence has been regarded as providing a simpler and more sensitive mode of detection in environmental sensing systems.
The phenomenon of bioluminescence first came under serious scientific scrutiny by Raphel Dubois in 1885 when he observed that the cell-free extracts obtained from the luminescent beetle Pyrophrus and the luminescent clam Pholas gave a light emitting reaction in vitro when mixed at room temperature. Since that time bioluminescent systems have been identified and examined in a myriad of different organisms including the common firefly, marine coelenterates, fish, terrestrial and freshwater worms, as well as bacterial, algal and fungal species.
Bacterial bioluminescence is phenomenon in which the products of 5 structural genes (luxA, luxB, luxC, luxD and luxE) work in concert to produce light. The luxD product generates a C14 fatty acid from a precursor. The C14 fatty acid is activated in an ATP dependent reaction to an acyl-enzyme conjugate through the action of the luxE product which couples bacterial bioluminescence to the cellular energetic state. The acyl-enzyme (luxE product) serves as a transfer agent, donating the acyl group to the luxC product. The acyl-LuxC binary complex is then reduced in a reaction in which NADPH serves as an electron pair and proton donor reducing the acyl conjugate to the C14 aldehyde. This reaction couples the reducing power of the cell to bacterial light emission. The light production reaction, catalyzed by luciferase (the product of luxA and luxB), generates light. The energy for light emission is provided by the aldehyde to fatty acid conversion and FMNH.sub.2 oxidation, providing another couple between light production and the cellular energy state.
Recently, naturally bioluminescent organisms have been used as the sensitive element in toxicity tests. The MICROTOX system, (Microbics Corp., Carlsbad, Calif.) is an example. The MICROTOX system measures the natural baseline luminescence of Photobacterium phosporeum and relates this to the hos
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LaRossa Robert Alan
Majarian William Robert
Van Dyk Tina Kangas
E. I. Du Pont de Nemours and Company
Guzo David
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