BACKGROUND Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. RESULTS Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. CONCLUSIONS Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.

中文翻译：

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大肠杆菌三重报告菌株的开发和表征，用于研究生物过程中的种群异质性。

背景技术如今，对高产量的健壮且成本有效的生物技术生产工艺的需求日益增长。尽管这些过程中的细胞起源于同基因培养，但是由内在和外在影响引起的异质性无处不在。为了加深对这种在机械上了解甚少的现象的理解，需要提供洞察单细胞生理的先进工具。结果基于工业相关生产宿主大肠杆菌BL21（DE3）及其修饰版本大肠杆菌T7E2，设计了两种大肠杆菌三重报告菌株。这些菌株携带三种不同的荧光蛋白，它们通过染色体整合在一起。在单个细胞生长之后，通过EmeraldGFP（EmGFP）-表达与核糖体启动子rrnB一起表达。单细胞的一般应激反应通​​过mStrawberry的σ因子rpoS的表达来监测，而nar-operon的表达与TagRFP657一起提供了单细胞的氧限制信息。首先，与野生型大肠杆菌BL21（DE3）相比，在间歇操作的搅拌罐式生物反应器中对菌株进行了表征。之后，在搅拌罐式生物反应器中，在不同的生长速率下响应于工业规模的葡萄糖和氧气扰动模拟梯度，在连续过程中证明了三重报告基因菌株在生物过程中研究种群异质性的适用性。监测群体和单细胞水平生理，评估三重报告基因菌株的荧光分布的一般生理和流式细胞仪分析。尽管两种三重报告菌株均反映了基于标记蛋白表达特征所预期的生理变化，但基于大肠杆菌T7E2的三重报告菌株对环境变化表现出更高的敏感性。对于这两种菌株，在从非生长阶段到生长阶段的过渡期都观察到了基因表达的噪音。显然，在某些过程条件下，例如分批培养的固定相，EmGFP和mStrawberry的荧光响应得以保留，而TagRFP657显示出明显的响应。结论可以使用本研究中开发的两个三重报告基因菌株追踪单细胞的生长，单细胞的一般应激反应和氧限制。