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

摘要

BackgroundMicrobial cell population heterogeneity is now recognized as a major source of issues in thedevelopment and optimization of bioprocesses. Even if single cell technologies are availablefor the study of microbial population heterogeneity, only a few of these methods are availablein order to study the dynamics of segregation directly in bioreactors. In this context, specificinterfaces have been developed in order to connect a flow cytometer directly to a bioreactorfor automated analyses. In this work, we propose a simplified version of such an interfaceand demonstrate its usefulness for multiplexed experiments.ResultsA low-cost automated flow cytometer has been used in order to monitor the synthesis of adestabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter andpropidium iodide (PI) uptake. The results obtained showed that the dynamics of GFPsynthesis are complex and can be attributed to a complex set of biological parameters, i.e. onthe one hand the release of protein into the extracellular medium and its uptake modifying theactivity 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 andthe stability of the reporter molecule.ConclusionThis work demonstrates that a simplified version of on-line FC can be used at the processlevel or in a multiplexed version to investigate the dynamics of complex physiologicalmechanisms. In this respect, the determination of new on-line parameters derived fromautomated FC is of primary importance in order to fully integrate the power of FC indedicated feedback control loops.