Assay for Detection and Enumeration of Genetically Engineered MicroorganismsWhich Is Based on the Activity of a Deregulated 2,4-Dichlorophenoxyacetate Monooxygenase

摘要

An assay system was developed for the enumeration of genetically engineeredmicroorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which coverts phenoxyacetate (PAA) to phenol. In PAA-amended cultures of Pseudomonas aeruginosa PAO1C(pRO103) and Pseudomonas putida PPO301(pRO103), strains which express a deregulated TFD monoxygenase, phenol production was proprtional to cell number. Phenol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotmetrically could detect as few as 10(3) cells per ml. The assay was corrobrated by monitoring the disappearnace of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography.