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

摘要

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. Among reporter proteins, the green fluorescent protein (GFP) is available in various mutants with different spectral characteristics. 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. In a previous study, we developed a bacterial biosensor for L-arabinose (L-ara) by employing E.coli cells harboring the plasmid pBAD-GFPuv, which contains the gene gfpuv (coding for GFPuv, a variant of GFP) and the system that activates its expression (the O/P region P_(BAD) and the regulatory protein AraC) in the presence of L-ara. In the present study, we modified pBAD-GFPuv by inserting the gene eyfp (coding for EYFP,, a variant of GFP with altered fluorescence spectra) its expression being activated by the P_(LAC) O/P region in the presence of isopropyl-β-D-thiogalacto pyranoside (IPTG). E. coli cells transformed with the plasmid therefore contain two reporter genes, which can be independently expressed and measured. The first reporter, GFPuv, expressed in response to the concentration of the analyte (L-ara), provides the analytical signal; the second reporter, EYFP, expressed at a constant level in the presence of a constant amount of IPTG can be used as an internal reference, allowing correction of the analytical signal measured from the first reporter. In fact, whenever GFPuv expression is altered because of ambient variations, EYFP expresion, that in standard conditions is constant, is altered in the same fashion. Therefore, just by performing two sequential fluorescence measurements, one can take into account the overall influence of non-specific stimuli on gene expression and accordingly correct the analytical signal.