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

Microbial Cell Factories

Research

Shanshan Han¹  Søren J Sørensen¹  Philippe Thonart²  Frank Delvigne²  Alison Brognaux³  Frédéric Lebeau⁴ 

[1] Section for microbiology, Department of Biology, University of Copenhagen, Universitetsparken, 15, Bygning, 1, 2100, Copenhagen, Denmark;Unité de Bio-industries/CWBI, Gembloux Agro-Bio Tech, Université de Liège, Passage des Déportés 2, 5030, Gembloux, Belgium;Unité de Bio-industries/CWBI, Gembloux Agro-Bio Tech, Université de Liège, Passage des Déportés 2, 5030, Gembloux, Belgium;Fond de la recherche scientifique (FRS-FNRS), Rue d’Egmont 5, 1000, Bruxelles, Belgium;Unité de mécanique et construction, Gembloux Agro-Bio Tech, University of Liège, Passage des Déportés 2, 5030, Gembloux, Belgium;

关键词: Automated flow cytometry;    Phenotypic heterogeneity;    Escherichia coli;    Membrane permeability;    Green fluorescent protein;    Mini-bioreactors;   

DOI  :  10.1186/1475-2859-12-100

 received in 2013-03-14, accepted in 2013-10-30,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundMicrobial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the study of microbial population heterogeneity, only a few of these methods are available in order to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect a flow cytometer directly to a bioreactor for automated analyses. In this work, we propose a simplified version of such an interface and demonstrate its usefulness for multiplexed experiments.ResultsA low-cost automated flow cytometer has been used in order to monitor the synthesis of a destabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter and propidium iodide (PI) uptake. The results obtained showed that the dynamics of GFP synthesis are complex and can be attributed to a complex set of biological parameters, i.e. on the one hand the release of protein into the extracellular medium and its uptake modifying the activity of the fis promoter, and on the other hand the stability of the GFP molecule itself, which can be attributed to the protease content and energy status of the cells. In this respect, multiplexed experiments have shown a correlation between heat shock and ATP content and the stability of the reporter molecule.ConclusionThis work demonstrates that a simplified version of on-line FC can be used at the process level or in a multiplexed version to investigate the dynamics of complex physiological mechanisms. In this respect, the determination of new on-line parameters derived from automated FC is of primary importance in order to fully integrate the power of FC in dedicated feedback control loops.

【 授权许可】

Unknown   
© Brognaux et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.