Monitoring and identifying genetically-engineered microorganisms in the environment by time-resolved laser fluorometry
A large percentage of the applications of Genetically Engineered Microorganisms (GEMs) involve their release into the environment. At the present time there is no rapid analytical method that can accurately identify and quantify the number of microorganisms and their foreign genes. In the past several years the author's laboratory has used successfully laser-based enzymatic assays to identify and differentiate pathogens, microorganisms, and genetically modified microorganisms. This work focused on the use of the above technology to track and identify agricultural beneficially GEMs that have been released into the environment. The first stage of this work dealt with the detection of the marker gene, the lactose operon. It was successfully demonstrated that the laser-based enzymatic assay can detect enzymatic activity in E. coli after 5 minutes of induction. Moreover, the author has achieved quantitation of GEMs in the laboratory down to 10[sup 4] cells with only a 30 minute incubation time. The second stage of this work dealt with the characterization of the analytical blank present in environmental samples. Strategies were devised to circumvent this interference and new substrates were synthesized that improved the S/B of the analysis. The last stage of this research dealt with devising new instrumental methods to detect small number (single cell) of microorganisms. These included incorporation of time-resolved detection in flow cytometry, Capillary Electrophoresis of microorganisms, and two-photon spectroscopy of centrosymmetric probes. The results found here will complement the large array of techniques available for monitoring and identifying GEMs in the environment. Ultimately, the technique chosen will depend heavily on the type of gene being monitored, the sensitivity required, and the environmental conditions.
- Research Organization:
- Purdue Univ., Lafayette, IN (United States)
- OSTI ID:
- 7114435
- Resource Relation:
- Other Information: Thesis (Ph.D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
GENE OPERONS
ELECTROPHORESIS
ENZYME IMMUNOASSAY
FLUORESCENCE SPECTROSCOPY
MICROORGANISMS
GENETIC ENGINEERING
MONITORING
CELL FLOW SYSTEMS
BIOASSAY
BIOTECHNOLOGY
EMISSION SPECTROSCOPY
IMMUNOASSAY
SPECTROSCOPY
400102* - Chemical & Spectral Procedures
550400 - Genetics