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Author Atlas, R. M. ♦ Bej, A. K. ♦ Steffan, R. J. ♦ Perlin, M. H.
Source United States Department of Energy Office of Scientific and Technical Information
Content type Text
Language English
Subject Keyword BASIC BIOLOGICAL SCIENCES ♦ ESCHERICHIA COLI ♦ GENETIC ENGINEERING ♦ DNA ♦ DNA HYBRIDIZATION ♦ GENE AMPLIFICATION ♦ GENE OPERONS ♦ GENES ♦ GROWTH ♦ MICROCOSMS ♦ PLASMIDS ♦ SOILS ♦ BACTERIA ♦ CELL CONSTITUENTS ♦ HYBRIDIZATION ♦ MICROORGANISMS ♦ NUCLEIC ACIDS ♦ ORGANIC COMPOUNDS ♦ Biochemistry
Abstract We have examined methods for tracking and limiting the survival capacity of genetically engineered microorganisms (GEMs) in the environment. By coupling efficient DNA extraction methodology with the poly-merase chain reaction (PCR) for the in vitro amplification of a gene probe target, we were able to detect 1 cell of a target organism/g sample by dot-blot hybridization against a background of 10{sup 11} diverse organisms. This is at least three orders of magnitude better sensitivity than has been achieved by previous gene probe methods. We have also constructed a model conditional-lethal plasmid vector (pBAP19h) for containing GEMs in the environment by placing the host killing gene (hok) from pPR633 under the control of the lac promoter and operator of plasmid pTZ19u in E. coli. In in vitro experiments, cultures inoculated with E. coli JM101 containing the conditional lethal plasmid pBAP19h ceased to grow 1-2 h after addition of IPTG, but returned to exponential growth after an additional 2 h of incubation; however, cells isolated 4 h after addition of IPTG no longer contained pBAP19h. When carbenicillin was added as a selective pressure for maintaining the plasmid, the suicide plasmid failed and after an initial period of cell decline, pBAP19h-bearing cells that had developed Hok resistance grew exponentially. In soil microcosm studies, the addition of IPTG resulted in a 90% decline in cells containing pBAP19h within 24 h of Hok induction and there was no evidence of renewed growth of hok-containing cells.
ISSN 08825696
Educational Use Research
Learning Resource Type Article
Publisher Place United States
Journal Hazardous Waste and Hazardous Materials
Volume Number 6
Issue Number 2


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