Design of growth-dependent biosensors based on destabilized GFP for the detection of physiological behavior of Escherichia coli in heterogeneous bioreactors

In this work we present the design and characterization of GFP-based reporter systems designed in order to describe cellular activity in ‘complex’, heterogeneous bioreactors. The reporter systems consist of Escherichia coli strains carrying growth dependent promoters fused to genes expressing stable and unstable variants of GFP, respectively. The response of Escherichia coli cells to transient exposure to glucose was studied in a two-compartment scale down bioreactor (SDR) consisting of a stirred tank reactor (STR) connected to plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation that often encountered in large-scale, fed-batch bioreactors. The response of E. coli coli to oxygen-poor and glucose-rich regions was simulated by continuously pumping E. coli cells from STR to the PFR. A concentrated glucose pulse (400 g/L) was consecutively added at the entrance of the PFR and samples were taken from PFR. The GFP expressions were significantly marked after 10 hours of culture in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat (D=0.14 h-1) with programmed glucose perturbation (30 g/L, frequency: 100/900 s) suggested that the activities of the promoter are linked with the substrate limitation signal. Taken together with immunoblot analysis, we suppose protein leakage is responsible for the overexpression of fis and the related promoters, such as rrnB in this case study, but additional works are required in order to confirm this relationship. Our finding are of great interest for industrial application since the GFP signal can be detected very early during the culture and is related to relevant physiological changes. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors.