MONITORING A GENETICALLY ENGINEERED BACTERIUM IN A FRESH-WATER ENVIRONMENT BY RAPID ENZYMATIC AMPLIFICATION OF A SYNTHETIC DNA NUMBER-PLATE

In order to set up a sensitive and reliable detection method to monitor environmentally released genetically engineered microorganisms (GEMs) a 72-bp, double-stranded DNA fragment has been built by annealing and ligating four synthetic oligonucleotides. Binding sites for two 20-mer oligonucleotides are situated inside the DNA fragment, flanking the centre. Into the central part of the construction a 30-nucleotide identification sequence has been fitted. Thanks to the presence of the two oligonucleotide binding sites, the synthetic construction (''number-plate'') can be submitted to enzymatic amplification using the polymerase chain reaction (PCR), thus enabling the identification system to take advantage of the outstanding sensitivity of this technique. When released into a freshwater microcosm, cells of Pseudomonas putida carrying a ''number-plated'' chromosome could be easily and rapidly detected merely by submitting boiled cell sediments to PCR amplification.