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Environmental Monitoring by Fluorescence Induction and Detection

Objective



Objective : Novel environmental biosensors are to be developed which detect and report the presence of genotoxic and other stress inducing chemicals in water.

The special expertise of the partners both in Molecular Genetics and in Instrument Development will be exploited in the development of genetically engineered sensors of damage-induced gene expression which can be monitored using solid and liquid phase detector systems.
The promoter of the DNA damage induced gene RAD54, the DNA-damage Response Element (DRE) and the Stress Response Element (STRE) in the budding yeast Saccharomyces cerevisiae will be linked to genes encoding variants of the jellyfish Green Fluorescent Protein (GFP). These yeasts will form the living component of biosensors. The measurable objectives of this work will be:
- Increased speed and sensitivity in damage-induced repair assays. - Development of novel automated, high through-put damage-detection technology.
- Proof of principal in screening for chemicals which affect repair in both model compounds and environmentally derived samples.

Novelty : Our approach exploits two aspects of novelty which represent advances in the state of the art of environmental sensing:

- The use of stress-inducible eukaryotic promoters to modulate induction of Green Fluorescent Protein - a harmless and simply estimated reporter. - Assessment of the activity DNA damage repair systems. This is an alternative to the assessment of lasting DNA damage in "Ames" tests.
Context :

Catastrophic changes to a water supply are easily detected - either by failures of industrial plant or water treatment facilities or by the sudden death of native species. This proposal does not intend to provide early warning where such obvious indicators exist. Instead it is hoped to detect longer term changes such as those arising for the leaching of waters from domestic/industrial landfill or increases in accidental or deliberate sublethal discharge where contamination is not immediately obvious. In these situations continuous remote sensing devices or continuous delivery of samples from the water system to a testing facility is required. Most DNA damage is seemlessly repaired.
As a consequence those methods, including descendants of the Ames Test, which assay lasting damage either in the form of misrepaired DNA (mutations and recombination) or unrepaired damage in the form of fragmented DNA underestimate the amount of DNA damage happening. Furthermore Bacterial genetics and biochemistry differs from that of the eukaryotes (from yeasts to humans) and the different processing of damage and activation of chemicals once inside a bacterial cell can contributes to false positive or negative results. We are proposing to focus our efforts on the assessment of repair activity, the events that happen before such genetic endpoints might develop. We have chosen yeast as a model because, amongst the eukaryotes, it has the best characterised repair system. In addition, the intrinsic problems of bacteria should be avoided. A RAD54-GFP reporter has already been constructed and tested in a pilot programme linking 2 partners.
Content : There will be 2 parallel programmes of activity.

Molecular Biological Development. Three new types of stress reporter will be constructed to exploit the new technologies:
(a) improved RAD54 reporters
(b) new DNA damage induced reporters
(c) an alternative stress reporter.

Technological Development. Three different GFP assessment technologies will be designed to suit different applications:
(a) Microtitre plate assayj
(b)Miniaturized irnmobilised-cell assay,
(c) Multi-channel Microfabricated Fluorescence-Activated Cell Sorter.

Coordinator

University of Manchester Institute of Science and Technology (UMIST)
Address
Sackville Street
M60 1QD Manchester
United Kingdom

Participants (2)

HANS KNOELL INSTITUTE FOR NATURAL PRODUCTS RESEARCH
Germany
Address
11,Beutenbergstrasse 11
07745 Jena
Universität Bern
Switzerland
Address
5,Sidlerstraße
3012 Bern