Reducing phenotypic instabilities of microbial population during continuous cultivation based on cell switching dynamics

Abstract

Predicting the fate of a microbial population (i.e., growth, gene expression…) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation processes. Indeed, the dynamic nature of continuous cultivation process implies the potential deviation of the microbial population involving genotypic and phenotypic diversification. This work has been focused on the induction of the arabinose operon in Escherichia coli as a model system. As a preliminary step, the GFP level triggered by an arabinose-inducible PBAD promoter has been tracked by flow cytometry in chemostat with glucose-arabinose co-feeding. Ampicillin was used as an “unstable” selective marker, allowing the simultaneous investigation of the effect of phenotypic diversification and genetic instability in continuous cultures. Under classical chemostat operation, the system was very unstable, with only a small fraction of cells (less than 10%) being able to accumulate GFP to a large extent, this fraction rapidly collapsing with time and going below 10% of the total population. On the long run, this phenotypic diversification was followed by an extensive loss of plasmid. In a second set of experiments, continuous cultivation was performed by adding either glucose or arabinose, based on the ability of individual cells for switching from low GFP to high GFP states, according to a technology called segregostat. In segregostat mode of cultivation, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the stochastic switching capabilities of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to a prolonged maintenance of the induction level with limited impact of phenotypic diversification and genetic instability for more than 68 generations