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ASSAY FOR DETECTION AD ENUMERATION OF GENETICALLY ENGINEERED MICROORGANISMS WHICH IS BASED ON THE ACTIVITY OF A DEREGULATED 2,4-DICHLOROPHENOXYACETATE MONOOXYGENASE
Citation:
King, R., K. Short, AND R. Seidler. ASSAY FOR DETECTION AD ENUMERATION OF GENETICALLY ENGINEERED MICROORGANISMS WHICH IS BASED ON THE ACTIVITY OF A DEREGULATED 2,4-DICHLOROPHENOXYACETATE MONOOXYGENASE. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-94/469 (NTIS PB95137311), 1991.
Description:
An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which coverts phenoxyacetate (PAA) to phenol. n PAA-amended cultures of Pseudomonas aeruginosa PAO1C (pRO103) and Pseudomonas putida PPO301 (pRO103), strains which express a deregulated TFD monoxygenase, phenol production was proportional to cell number. henol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotometrically could detect as few as 10 3 cells per ml. he assay was corroborated by monitoring the disappearance of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography. he AAPPC assay was modified for use with plate cultures and clearly distinguished colonies of PPO301 (pRO103) and PAO1C (pRO103) from a strain expressing a regulated TFD monooxygenase. olonies of P. putida PPO301( pRO101) remained cream colored, while colonies of PPO301 (pRO103) and PAOIC (pRO103) turned a distinct red.