Engineering whole-cell biosensors to evaluate the effect of osmotic conditions on bacteria

Enhanced green fluorescent protein (eGFP) is a variant of wild-type GFP humanized for optimal expression in mammalian cell lines. A computational approach comparing wtGFP and eGFP showed the occurrence of rare proline codons within the eGFP gene that could interfere with and hamper protein production in prokaryotic expression systems. The eGFP gene excised from mammalian plasmid pEGFP N3 was used for construction of two inducible promoter-reporter fusions, T7-eGFP and P-proU-eGFP, through directional cloning. The T7-eGFP fusion confirmed expression of eGFP protein within the bacterial strain, showing a fluorescent green cell pellet and overexpression of the similar to 29 kDa eGFP protein upon induction with IPTG. The proU operon aids in osmoadaptation by encoding a transport system for uptake of various compatible solutes, including glycine-betaine and proline. Expression of the proU operon is induced upon growth of bacteria in media of elevated osmolarity. When coupled to an eGFP reporter, a time course study using fluorometry demonstrated that induction of P-proU in Escherichia coli occurred rapidly. The P-proU induction and recombinant eGFP production depends on time and concentration of solute (NaCl) in the medium. Cells containing the P-proU-eGFP fusion showed maximum promoter activity at 500 mM concentration of NaCl with a sensitivity of the P-proU promoter being 50 mM. The relative fluorescence reflected the amount of protein synthesized proportional to the activity of induced promoter and effect of NaCl on growth was also taken into consideration. Thus, such environmentally regulated highly sensitive promoters with enhanced reporters could possibly be used as whole-cell biosensors