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A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria

Yan Y. Goh,^1, Bow Ho,² and Jeak L. Ding^1*

Department of Biological Sciences,¹ Department of Microbiology, National University of Singapore, Singapore 117543²

Received 22 April 2002/ Accepted 5 September 2002

Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFP_i) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFP_i proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFP_i G10 and G12 to lipid A were established. The K_D values of 7.16 x 10^-7 M for G10 and 8.15 x 10^-8 M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFP_i biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFP_i specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFP_i in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

Present address: Temasek Applied Science School, Temasek Polytechnic, Singapore 529757.