DEVELOPMENT AND CHARACTERIZATION OF A FLUORESCENT WHOLE-CELL BIOSENSOR FOR L-ARABINOSE WITH INTERNAL RESPONSE CORRECTION USING TWO GFP MUTANTS

Genetically engineered bacterial biosensors are obtained by introducing into a cell a reporter gene the expression of which is activated as a consequence of the interaction between the analyte and an operator/promoter (O/P) sequence, usually with the intervention of a regulatory protein.¹ Among reporter proteins, the green fluorescent protein (GFP) is available in various mutants with different spectral characteristics.² One main drawback encountered in the analytical application to real samples of such sensing devices is the non-specific effect of matrix components on the whole-cell biosensor response. In fact, being bacteria (complex biosystems) where hundreds of metabolic pathways influence one another, the modulation of the expression of the reporter protein and the related fluorescence is the result not only of the specific interaction with the analyte, but also of the overall metabolic activity of the cell. This requires the use of several controls for assessing bacterial viability and accordingly correct the analytical signal. In this context, the use of an internal reference signal control is highly desirable since it allows correcting the response, thus “cleaning” the analytical signal from non-specific interferences…