Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-09-15
1998-07-07
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
4353201, 4352523, 435 691, 536 231, C12Q 168, C07H 2104, C12N 1563
Patent
active
057766819
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a biotest in which the metal content is determined qualitatively and/or quantitatively from a liquid, gaseous, or solid sample by means of genetically manipulated cells.
Methods in which living cells or organisms are used as tools are in general called biotests. Many of the biotests developed utilize bacterial or yeast cells. A great deal of hope and interest have been placed on the use of microbes as rapid tests for heavy-metal residues. Since microbiological methods utilize bacteria or spores of bacteria, the sensitivity of the test bacterium to a heavy metal is of crucial importance in these methods. Up to the present it has been necessary to make compromises in the selection of suitable test microbes, since, for example, high sensitivity to heavy metals and other properties required of a test microbe have not necessarily been properties of one and the same bacterial strain.
The use of microbes in testing for heavy-metal residues is limited above all by the slow performance and insensitivity of the methods. Since the methods in one way or another always control the growth of the test microbes, it is inconceivable that a test could be performed in a time shorter than one hour. This is due to the fact that, even at its most rapid, microbial growth is slow. Furthermore, in a number of tests the microbes are freeze-dried microbes or spores; this slows down the performing of the tests even more. The present-day microbiological heavy-metal tests are not capable of determining single heavy metals or their groups; instead, they detect all toxic heavy metals to which the microbe concerned is sensitive.
Determination methods which are based on the measuring of bioluminescence, i.e. the production of light, have also been developed. In seas there are, living as planktonic populations or in a symbiosis with fish, a number of different bacterial strains which produce light as a byproduct of their metabolism. The production of light is accounted for by an enzyme called luciferase, the chemical reaction of its catalysis being as follows: and oxygen, forming the corresponding oxidized products and light at a wavelength of 490 nm. The optimum temperature for bacterial luciferase is in general approx. 25.degree. C., and it is very rapidly inactivated at the physiological temperature 37.degree. C.
A method (trade name Microtox.TM.), available even commercially and utilizing Photobacterium fischeri bacteria measures the overall toxicity of the environment (Bulich and Greene, 1979, International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. State Printing and Publishing, Westlake Village, Calif., pp. 193-211). The said bacterial cells used in the determination have been freeze-dried, and after rehydration they can be used for measuring, among other things, heavy-metal residues in general at a micromolar sensitivity level simply by measuring changes in the intensity of light, which changes correlate with the toxicity of the environment. The sample to be investigated and the bacteria are incubated together, and the presence of toxic substances is detected from the lowered luminescence level of the bacteria as compared with a control sample. The method has a number of drawbacks, the worst perhaps being a continuous and rapid luminescence decrease, which is in general compensated for in the test by using apparatus technology. This results in very expensive apparatus. The method also requires high salinity (3%), which has been shown to reduce the toxic effect of certain substances. The method is also nonspecific, and it reacts in the same manner to a number of heavy metals and other environmental toxins.
The gene mechanisms responsible for luminescence properties can also be transferred into other organisms by modern genetic engineering techniques, and thus a previously dark cell can be caused to emit light in the manner of the original bioluminescent bacterial cell (Belas et al., Science, 218, 791-793). These methods utilize luciferase genes isolated from luminescent P.
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Karp Matti
Virta Marko
Atzel Amy
Jones W. Gary
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