Fluorescence-PCR Assays and Isolation of Luminescent Bacterial Clones Using an Automated Plate Reader

The genes responsible for luminescence in various species of the marine microorganism Photobacterium, have been used for many years as a tool by researchers and instructors. In particular, the lux operon of Photobacterium fischeri has been used by many instructors to teach recombinant DNA techniques. Two methods using an automated plate reader and multiwell plates were applied to a set of previously-published exercises. In these exercises that involve transfer of lux genes to Escherichia coli to create a luminescent phenotype, this technology was used to screen for Lux(+) colonies. It was found to be more convenient and more sensitive than the previously used method; that is, assaying bacterial plates by direct observation. Eight students synthesized four genomic libraries and isolated six Lux(+) clones. The fluorescent-detection feature of the plate reader was used to verify amplification of target sequence in polymerase chain reaction (PCR) reactions. Lux(+) E. coli colony lysates were examined. An exonuclease-activated, fluorescent DNA probe generated a signal on hybridization to an amplified portion of the luxA gene in each lysate tested. This method is suggested as a means of demonstrating the concept of real-time PCR without the expense of the specialized device typically used for this technique.