Biology subjects

Zicler, A.

Publications and source records attributed to Zicler, A..

4 recordsLinked to original sources

bioRxiv2024

Lowering the switching cost related to the activation of burdensome gene circuits promotes cell population homogeneity and productivity

The activation of gene circuits can impose a significant burden on cells, leading to heterogeneous expression and reduced productivity. In this work, we focused on the T7 production system in E. coli BL21, a prime example of a burdensome gene circuit, to investigate the main cause for this gene expression heterogeneity and methods to mitigate it. Based on continuous cultivation analyzed and control by automated flow cytometry, we quantified the trade-off between cellular growth and gene expression and tracked the cell-to-cell heterogeneity in gene expression (measured as entropy). We concluded that the growth reduction associated to the activation of the burdensome gene circuit, i.e., the switching cost, is at the origin of the population heterogeneity. The loss of growth rate imposed by the burdensome activation of the gene is compensated at the population level by the overgrowth of less induced cells that safeguard the population by generating entropy. We tried to homogenize the population by pulsing the inducer with increasing frequency but found that the population escapes control through promoter mutation, leading to a genotype exhibiting reduced gene expression, but also, reduced entropy. To engineer a more homogeneous population without sacrificing gene expression, we decreased the switching cost associated to the induction by lowering the quality of the main carbon source. This strategy successfully led to a more homogeneous and productive population. Our approach allows for a precise quantification of the trade-off between growth and gene expression in cell population cultivated under dynamic conditions and highlights the importance of the switching cost for designing efficient approaches of cell population control.

systems biology↗

bioRxiv2024

Automated adjustment of metabolic niches enables the control of natural and engineered microbial co-cultures

A lot of attention has been given to the understanding of microbial interactions leading to stable co-cultures, but the resulting technologies have been rarely challenged in dynamic cultivation conditions. In this work, substrate pulsing was performed to promote better control of the metabolic niches corresponding to each species, leading to the continuous co-cultivation of diverse microbial organisms. For this purpose, we used a cell-machine interface relying on automated flow cytometry, allowing to adjust the temporal profile of two metabolic niches according to a rhythm ensuring the successive growth of two species i.e., in our case a yeast and a bacterium. The resulting approach, called Automated Adjustment of Metabolic Niches (AAMN), was successfully employed for stabilizing both cooperative and competitive co-cultures. Additionally, AAMN can be considered as an enabling technology for the deployment of co-cultures in bioprocesses, demonstrated here based on the continuous bioproduction of p-coumaric acid. Taken altogether, the data accumulated suggest that AAMN could be used for a wider range of biological systems, but also to gain fundamental insights about microbial interaction mechanisms.

microbiology↗

bioRxiv2023

Fitness cost associated with cell phenotypic switching drives population diversification dynamics and controllability

Isogenic cell populations can cope with stress conditions by switching to alternative phenotypes. Even if it can lead to increased fitness in a natural context, this feature is typically unwanted for a range of applications (e.g., bioproduction, synthetic biology, biomedicine...) where it tends to decrease the controllability of the cellular response. However, little is known about the diversification profiles that can be adopted by a cell population. We characterized the diversification dynamics for various systems (bacteria and yeast) and for different phenotypes (utilization of alternative carbon sources, general stress response and more complex development patterns). Interestingly, our results suggest that the diversification dynamics and the fitness cost associated with cell switching are coupled. For quantifying the contribution of the switching cost on population dynamics, we built a stochastic model that allowed us to reproduce the dynamics observed experimentally and identified three diversification regimes, i.e., constrained (at low switching cost), dispersed (at medium and high switching cost), and bursty (for very high switching cost). Furthermore, we used a cell-machine interface that we call the Segregostat to demonstrate that different levels of control can be applied to these diversification regimes, enabling applications involving more precise cellular responses.

microbiology↗

bioRxiv2021

Reducing phenotypic and genotypic instabilities of microbial population during continuous cultivation based on stochastic switching dynamics

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. 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 ParaBAD promoter has been tracked by flow cytometry in chemostat with glucose-arabinose co-feeding. For a large range of glucose-arabinose co-feeding, the simultaneous occurrence of GFP positive and negative subpopulation was observed. 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 phenotypic 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 on phenotypic diversity for more than 60 generations. This result suggests that constraining individual cells into a given phenotypic trajectory is maybe not the best strategy for directing cell population. Instead, allowing individual cells switching around a predefined threshold seems to be a robust strategy leading to oscillating, but predictable, cell population behavior.

microbiology↗