Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-09
2002-06-18
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S189000, C435S254200, C435S254210
Reexamination Certificate
active
06406856
ABSTRACT:
The present invention relates to a eukaryotic biosensor for the detection of xenobiotics and bioactive compounds. It will be appreciated that xenobiotics are not necessarily bioactive.
The problems of environmental contamination with toxic chemicals are becoming increasingly apparent. Such pollution has fuelled the need to develop novel, rapid and inexpensive methods for toxin detection in the environment.
Assay systems such as High-Performance Liquid Chromatography accurately predict quantities of chemical components but will not indicate toxicity or bioavailability (potential of compound to react with cellular components).
Prokaryotic biosensors, for example MICROTOX®, were created to indicate the toxicity and bioavailability of chemical components. Prokaryotic biosensors are unicellular living organisms that provide information about in vivo toxicity rapidly and reliably. They can detect a wide range of pollutants within a certain narrow pH range, whilst simultaneously assessing bioavailability in environmental samples. However, the MICROTOX® biosensors are generally not sensitive to eukaryotic-specific molecules, such as DIURON® 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and they only operate within a narrow pH range.
Existing assays for quantifying in vivo toxicity of chemicals for eukaryotic cells exploit whole animal models or tissue culture, which is both time consuming and expensive. The unicellular yeast
Saccharomyces cerevisiae
has previously been shown to function as a biosensor using BOD (Biochemical Oxygen Demand) respirometry. However, this BOD assay system is also time consuming and expensive.
The usual method for selecting plasmids or episomes which have undergone bioengineering is that during the genetic engineering procedure antibiotic resistance genes are transferred in the vector with the desired gene. Therefore when the transfer is successful the plasmid and therefore the host cell is resistant to antibiotics. The bioengineered cells can then be selected by growing them in the presence of antibiotics. However, antibiotic resistance is becoming more and more of a problem. There are now ‘Superbugs’ which cannot be treated by antibiotics and many other bacteria are resistant to all but one antibiotic. Now there is a concerted effort on the part of scientists to reduce the number of antibiotic resistence genes that they transfer from one microorganism to another.
One object of the present invention is to develop a cheap and quick assay for quantifying the in vivo toxicity of chemical components for eukaryotic cells which will operate over a wide pH range and function in the presence of an organic solvent.
Another object of the present invention is to develop a cheap and quick assay for quantifying the in vivo toxicity of chemical components for eukaryotic cells through chromosomal integration of the luciferase gene without the transfer of antibiotic resistance.
According to a first aspect of the invention there is provided a method for evaluating a biological effect of a substance, the method comprising the following steps:
a) preparing a eukaryotic biosensor engineered with a gene which constitutively expresses a light emitting protein;
b) sampling the substance;
c) subjecting the sampled substance at any pH between pH1 and pH12 to an assay in the presence of the biosensor; and
d) monitoring any changes in light output.
It should be noted that the term “biological effects” include all toxicity testing and the steps in the above identified method can be carried out in any order. In addition, a substance can be taken to be, inter alia, a liquid, such as water, a solid or suspension or colloid or sediment or sludge.
Typically biosensors have been produced from prokaryotic cells which can only operate in a narrow pH range. The inventive eukaryotic biosensor can function at any pH between pH1 and pH12 which makes it more useful for environmental samples and commercially viable. The biosensor is cheap and easy to produce so it can be used in mass and routine screening of water supplies. The pH tolerance of the yeast biosensor for example will enable toxicity assessment of industrial waste and toxicity in extreme environmental samples such as acid mining waste.
In a preferred embodiment the eukaryotic biosensor of the invention is derived from the Saccharomyces genus and preferably from
Saccharomyces cerevisiae. S. cerevisiae
is an ideal cell to function as a biosensor because it can tolerate an external pH within the range pH1 and pH12 and it is permeable to many xenobiotics and bioactive compounds. It also senses the toxic effect of the contaminated liquids on eukaryotic cells and therefore more accurately indicates the liquid samples possible toxicity to higher order organisms, and particularly to mammals.
Conveniently the light emitting protein is a luciferase.
In a preferred embodiment the luciferase is either a bacterial luciferase or a eukaryotic luciferase. Preferably the bacterial luciferase is from
Vibrio harveyi
and the eukaryotic luciferase is a firefly luciferase from
Photinus pyralis.
Both the luciferases require an exogenous addition of the substrate n-decyl aldehyde and luciferin, respectively. The luciferin is an amphipathic molecule that has a carboxyl group charged at physiological pH which prevents its free passage across cell membranes. This problem is overcome by acidifying the sample containing the cells and the biosensor after exposure to the potentially toxic sample. It is therefore important that these cells can remain metabolically active at an acidic pH. Both these luciferase genes produce light emitting proteins that require energy from the eukaryotic cell to produce light and therefore the level of light output is dependant on the health of the cell. Thus if the cell viability is challenged by components of the sample, for example due to the presence of a toxin, the level of light will fall and the resultant toxic effect of the sample can be noted.
The substance may be contaminated with a xenobiotic compound or a bioactive compound. For example, a xenobiotic compound may be selected from copper, 3,5-dichlorophenol, 2,4-dichlorophenol, MECOPROP® (±)-2-(4-chloro-0-tolyloxy) propionic acid, DIURON®, paralytic shell fish toxins, benzo (a) pyrene and MCPA. Copper, 3,5-dichlorophenol and 2,4-dichlorophenol are compounds found in industrial waste, which can find their way into rivers and lakes through accidental or deliberate dumping. They can also leach out of the soil around industrial waste plants. They are toxic to river dwelling organisms and those higher up the food chain. Accordingly, their levels in river water must be carefully monitored. MECOPROP® and DIURON® are herbicides which are used liberally by farmers. They leach out of the soil into rivers where once again they and their biologically active derivatives are toxic to the river dwelling organisms and those higher up the food chain.
Organic solvents such as ethanol, methanol, acetone and DMSO are also harmful to organisms. The biosensor herein described in stable in an environment which contains such organic solvents and it can therefore be used to identify substances which are being tested for toxicity to higher organisms, in the presence of these solvents. It will be appreciated that the presence of these solvents thus does not detract from the assay of the substances.
In a second aspect of the invention there is provided a biosensor comprising a bioengineered organism from the Saccharomyces genus expressing a light emitting protein gene wherein the level of light emitted by the organisms is dependent on the environmental conditions surrounding the organism. A gene conferring antibiotic resistance is not necessarily required for biosensor selection. The light emitting protein gene may be present on a plasmid which has been transferred into Saccharomyces species.
In a third aspect of the invention there is provided a biosensor comprising a eukaryotic bioengineered organism with a chromosomally integrated gene fragment expressing a light emitting protein wherein t
Glover Lesley Anne
Hollis Roger Paul
Killham Kenneth Stuart
Aberdeen University
Ketter James
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