Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-07
2001-12-11
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007600, C435S007230, C435S008000, C435S189000, C435S287100, C435S252330, C435S325000, C435S007320, C435S007370, C536S023200, C536S024100
Reexamination Certificate
active
06329160
ABSTRACT:
TECHNICAL FIELD
The invention relates to the use of biosensors for detecting environmental signals based on the measurement of genetic responses of cells to those signals.
BACKGROUND ART
The standard approach for biosensor measurements “based on genetic responses” is to attach reporter genes to the relevant promoters and to measure the signal (which consists of the activity of the enzyme encoded by the reporter gene) in response to the analyte of interest. The first publication dealing with the use of the bacterial luciferase operon to achieve such measurements appeared in 1984 (Baldwin et al., 1984). Since that time many more publications detailing the use of this reporter to probe the activity of promoters have appeared. Today there are probably several thousand publications on this subject. Most of these publications rely on the use of luxAB fusions or of entire lux cassettes. The more restricted goal of measuring the concentration of pollutants with such constructs was first proposed and demonstrated by Gary Sayler's group at the University of Tennessee in the USA (King et al., 1990). Burlage & Kuo (1994) recently reviewed the application of such biosensors with respect to environmental monitoring applications.
Practically all of the constructs described utilise either the entire lux operon for activity measurement or the luciferase part of it (lux AB, in this case the activity is measured by external addition of the aldehyde substrate). The major disadvantage of using the entire lux operon is that production of the enzyme responsible for generating the substrate of luciferase (the fatty acid reductase encoded by lux CDE) occurs simultaneously with the synthesis of luciferase. It is therefore probable that the amount of substrate produced by the cell will be insufficient for saturation of luciferase. The present inventors have introduced modifications into constructs suitable for biosensors in an attempt to address this disadvantage thereby allowing maximal light output as soon as luciferase is synthesised.
DISCLOSURE OF THE INVENTION
In a first aspect, the present invention consists in a genetic construct for use in a biosensor comprising:
(a) a first nucleic acid molecule including a sequence encoding a reporter molecule having a detectable activity; and
(b) a second nucleic acid molecule including a sequence encoding an enzyme which produces a substrate for the reporter molecule, the first sequence being under the control of a first inducible promoter and the second sequence being under the control of a second inducible promoter.
In a preferred embodiment, the first nucleic acid molecule encodes bacterial luciferase Lux AB or a functional equivalent thereof and the second nucleic acid molecule encodes Lux CDE enzyme fatty acid reductase or a functional equivalent thereof. These genes may be obtained from any of the lux operons of bioluminescent microorganisms, most of which belong to the genera Vibrio, Xenorhabdus, Photorhabdus and Photobacterium (Meighen, 1994). The detectible activity in this system is the generation of light.
It will be appreciated that any inducible promoters will be suitable for the present invention. Examples of some promoters that are suitable are listed in Appendix 2. This list, however, is not an exhaustive list but is provided to demonstrate the large number of possible promoters. The choice of the first promoter is often dependent on the environmental signal to which the biosensor is adapted to react. In particular, when the biosensor is used to detect xylene, the Pu promoter is especially suitable as the first promoter.
A further preferred embodiment of the first aspect of the present invention is the genetic construct adapted for the detection of xylene set out in FIG.
14
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The distinct advantage of the genetic construct of the present invention is that when in a cell, expression of the enzyme can be induced separately from the expression of the reporter molecule. This allows the cell to be loaded with substrate produced by the enzyme. The substrate is immediately available for use by the reporter molecule when it is produced, thereby giving a quick response to the signal detected. When the reporter molecule is made in response to an environmental signal, it can react maximally to give a detectable activity. Preferably the first promoter is inducible by exposure to an environmental signal to be tested. Induction can be achieved directly by the signal or indirectly by activating one or more separate pathways in the cell containing the construct.
The genetic construct can include further nucleic acid molecules encoding auxiliary element or elements required for activation of the reporter molecule via its promoter. Examples is include regulatory genes or nucleic acid molecules expressing “second message” molecules to activate the first promoter.
The effector specificities of existing promoters which depend on an indirect activation pathway by regulatory genes can be altered by mutation of the signal binding site of the regulatory protein (Ramos et al.,
Proc. Natl. Acad. Sci. USA
, 83, 8467-8471, 1986). New promoters can be identified using promoterless promoter-probe vectors based on transposons such as those described by deLorenzo et al. (deLorenzo, V.; Herrero, M.; Jakubzik, U. & Timmis, K. N.
J. Bact
., 172, 6568-6572, 1990) or Sohaskey et al. (Sohaskey, C. D.; Im, H. & Schauer, A. T.,
J. Bact
., 1674, 367-376, 1992).
In a second aspect, the present invention consists in a biosensor for measuring an environmental signal comprising a cell including the genetic construct of the first aspect of the present invention and a means for measuring the activity of the reporter molecule in the cell when the cell has been exposed to the environmental signal.
The cell can be any cell including bacterial, yeast, fungal and other plant cells and animal. Preferably the cell is a bacterial cell. The environmental signal can be pollutants, toxins, temperature, irradiation, biological, and chemical.
When the bacterial luciferase system is used, the basic genetic units of the sensor are the second nucleic acid molecule encoding the production of the fatty acid reductase (unit 2), the first nucleic acid molecule encoding the reporter element containing the luciferase genes and the first promoter (unit 1) as well as in some instances a third unit which would supply auxiliary elements for activation of the sensor element such as regulatory genes (unit 3). In commercial sensors all elements would be ideally inserted in the chromosome of the host organism. Alternatively, all units may be placed on plasmids, or a combination of chromosomally inserted elements and plasmid-borne constructs may be used. For example, where three units are used, unit 2 and unit 3 may be inserted in the chromosome whilst unit 1 is located on a plasmid.
A functional biosensor unit using the bacterial luciferase system consists of a device where the sample to be tested is contacted with biosensor cells which include a genetic construct according to the first aspect of the present invention (in a cuvette for example) and an instrument capable of measuring the light output of the cells. Any light measuring device would in principle be applicable, but the most appropriate systems are photomultipliers, charge coupled devices, luminometers, photometers, fiber-optic cables or liquid scintillation counters. These systems can be portable or laboratory based, they can be adapted for single analysis or high throughput devices for multiple analyses. A survey of more than 90 commercially available systems suitable for the present invention has been published by Stanley in J. Bioluminesc. Chemiluminescence, 7, 77-108, 1992 and regular updates have appeared since.
Analytes can be measured by the biosensor when adsorbed to surfaces, dissolved in liquid media or when present in the gasphase. Condition for measurement is that sufficient moisture be available to ensure the function of the biosensor cells which include a genetic construct. Ideally, samples are diluted with a buffer of appropriate co
Jury Karen
Schneider Rene
Vancov Tony
Browdy and Neimark
CRC for Waste Management and Pollution Control Limited
Le Long V.
Pham Minh-Quan K.
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