Direct and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: Design of milliliter scale‐down bioreactors

Abstract

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e., uspA::gfp, csiE::gfp, and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response to substrate limitation and are significantly repressed when cultures are carried out in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini-scale-down platform is proposed as a high throughput tool to rapidly investigate the usefulness of a given microbial biosensor. This platform is composed of shake flasks able to operate in fed-batch mode either using the slow release or the intermittent feeding principle. Local heterogeneities were reproduced at the level of these mini-bioreactors (operating under the intermittent feeding principle) and caused a decrease in GFP expression as in conventional scale-down reactors. The presence of GFP in supernatants was also noted and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with membrane permeability.

A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

“…This phenomenon was unexpected since GFP would remain in steady-state if the activation of the fis promoter is effectively proportional to the growth rate, suggesting that additional physiological mechanisms are involved. In our previous studies, we have shown that proteins are secreted to the extracellular medium when microbial cells are exposed to prolonged substrate limitation [23,24]. This phenomenon has been reported by several authors and is generally termed protein leakage [25,26].…”

Heterogeneous oxygen availability affects the titer and topology but not the fidelity of plasmid DNA produced by Escherichia coli

“…This may affect the quality of the recombinant protein produced. In the present study, DOT heterogeneities were simulated by transitions in the stirring rate, (similar to previous studies by Brognaux et al [23], and Han et al, [24]) and were compared with cultures under constant oxygen availability. Specifically, we measured the plasmid production capabilities of E. coli in three scenarios in relation to the oxygen availability, aerobic, microaerobic and oscillatory DOT conditions.…”

Fermentation profile of Saccharomyces cerevisiae and Candida tropicalis as starter cultures on barley malt medium

“…Propidium iodide (PI) was used to determine membrane permeability. After staining with PI, cells with compromised membranes were detected by flow cytometry using the following settings: FSC E00, FL3 600 previously described by Brognaux et al (2013).…”