Detection of benzene derivatives by recombinant E-coli with Ps promoter and GFP as a reporter protein

The TOL plasmid of Pseudomonas putida mt-2 encodes a series of enzymes for the degradation of benzene derivatives. The expression of the enzymes is controlled by the regulating protein XylR, of which the promoter, known as Ps, is activated in the presence of benzene derivatives. Sensitive and convenient sensing of benzene derivatives has been developed by fusing a gene encoding a green fluorescent protein (GFP) into the downstream of the Ps promoter. In the plasmid pTS301-GFP, GFP as a reporter protein is transcribed by controlling the promoter's activation by Xy1R and benzene derivatives. In the present study, a recombinant Escherichia coli transformed with this plasmid was applied to the environmental sensing of benzene derivatives in water. The expression of GFP was confirmed in water containing 0.1 mM m-xylene and fluorescence intensity was quantified by microplate reader. The optimal temperature for GFP expression was 27 degreesC and the lowest detectable concentration of xylene derivatives 0.1 mM. Benzene derivatives having CH3- or Cl- or both in the side chain of the benzene ring showed particularly high fluorescence intensity. To conclude, transformants harboring pTS301-GFP constitute an adaptable system for easy sensing of small amounts of benzene derivative in water. (C) 2003 Elsevier Science B.V. All rights reserved.