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USE OF A NOVEL PLASMID TO MONITOR THE FATE OF A GENETICALLY ENGINEERED PSEUDOMONAS PUTIDA STRAIN
Citation:
Genthener, F., R. Campbell, AND P. Pritchard. USE OF A NOVEL PLASMID TO MONITOR THE FATE OF A GENETICALLY ENGINEERED PSEUDOMONAS PUTIDA STRAIN. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-93/066 (NTIS PB93169001).
Description:
Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered microorganism (GEM) and its recombinant DNA in environmental samples. his broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin), and a fragment of eukaryotic DNA. he clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomanas putida RC-4, to grow on 3-chlorobenzoate. his catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, freeliving in the water column or when irreversibly bound to surfaces. he eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. n flow-through microcosms, RC-4(pSI30), undetectable as freeliving calls, was found by enrichment as irreversibly bound sessile forms. hese experiments revealed the stability of pSI30 and its utility in a "combination" detection system for tracking the survival of a GEM and its DNA in environmental samples.
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USE OF A NOVEL PLASMID TO MONITOR THE FATE OF A GENETICALLY ENGINEERED PSEUDOMONAS PUTIDA STRAIN