Unité de Bio-industries/CWBI, Gembloux Agro-Bio Tech, Université de Liège, Passage des Déportés 2, Gembloux 5030, Belgium.
Microb Cell Fact. 2013 Oct 31;12:100. doi: 10.1186/1475-2859-12-100.
Microbial 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.
A 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.
This 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.
微生物细胞群体异质性现在被认为是生物工艺开发和优化过程中出现问题的主要原因。即使有用于研究微生物群体异质性的单细胞技术，但为了直接在生物反应器中研究分相动力学，可用的方法也很少。在这种情况下，已经开发了特定的接口，以便将流式细胞仪直接连接到生物反应器，以进行自动化分析。在这项工作中，我们提出了这种接口的简化版本，并证明了其在多路复用实验中的有用性。
使用低成本自动化流式细胞仪来监测不稳定绿色荧光蛋白（GFP）在 fis 启动子和碘化丙啶（PI）摄取调控下的合成。结果表明，GFP 合成的动力学很复杂，可以归因于一组复杂的生物学参数，即一方面是蛋白质释放到细胞外培养基中及其摄取会改变 fis 启动子的活性，另一方面是 GFP 分子本身的稳定性，这归因于蛋白酶含量和细胞的能量状态。在这方面，多路复用实验表明热休克和 ATP 含量与报告分子的稳定性之间存在相关性。
这项工作表明，在线 FC 的简化版本可以在工艺水平或多路复用版本中用于研究复杂生理机制的动力学。在这方面，从自动 FC 得出的新在线参数的确定对于充分整合 FC 在专用反馈控制回路中的功能至关重要。
