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

ABSTRACT

In this work, we present the design and characterization of Green Fluorescent Protein (GFP)-based reporter systems designed 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 well-stirred tank reactor (STR) connected to a plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation of high glucose concentration and oxygen limitation that often encountered in large-scale, fed-batch bioreactors and the response of E. coli was simulated by continuously pumping microbial cells from STR to the PFR. We found that repeated addition of concentrated glucose pulses with varied frequency at the entrance of the PFR had consequences on strain physiological behavior. The GFP expressions were significantly marked after 10 h of cultivation in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat with programmed glucose perturbation suggested that the activities of the promoters were 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. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors.