Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems.
Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems.
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DOI：
10.1186/1475-2859-13-53
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发表时间：
2014-04-11
影响因子：
6.4
通讯作者：
Büchs J
中科院分区：
文献类型：
--
作者：
Kunze M;Roth S;Gartz E;Büchs J
用于微生物系统的新型高通量筛选系统，例如BioLector 技术操作简单，并提供多种光学在线测量选项。微量滴定板的并行化和小规模允许经济的高通量，因此可以在合理的时间内筛选许多参数。荧光蛋白作为荧光标签使得体内细胞蛋白的追踪成为一项常规任务。所有这些工具都极大地促进了对生物过程的理解。但是，存在一些陷阱可能会误导此类技术的用户。在这项工作中，对表达重组荧光蛋白 GFP、YFP、FbFP 和 mCherry 的大肠杆菌和酵母乳酸克鲁维酵母进行了研究。使用带有用于在 BioLector 系统中测量溶解氧张力 (DOT) 和 pH 值的光极的特殊微量滴定板进行培养。通过这种方式，通过光学信号在线监测微生物生长、蛋白质形成、DOT 和 pH 值。在这些研究中，很明显荧光蛋白会干扰光信号，导致错误的结果。在这项工作中，详细描述了这些影响，并提出了如何通过数学程序或测量方法的修改来纠正或最小化这些不利影响的可能性。此外，研究表明细胞形态的变化会影响散射光在线监测的生物量。这里报道的现象是指生物技术实验室的典型实验。因此，本工作强调了这些方面，以使 BioLector 等有价值技术的操作员意识到潜在的陷阱和由此产生的误解。通过正确的方法，可以最大程度地减少现有问题并加以解决。
New high-throughput screening systems for microbial systems, e.g. the BioLector technology, are simple to handle and offer various options of optical online measurements. The parallelization and small scale in microtiter plates allow economical high throughput and, hence, to screen many parameters in reasonable time. Fluorescent proteins as fluorescent tags made the tracking of cellular proteins in-vivo a routine task. All these tools significantly contribute to the understanding of bioprocesses. But, there are some pitfalls which might mislead the user of such techniques. In this work the bacterium E. coli and the yeast K. lactis expressing the recombinant fluorescent proteins GFP, YFP, FbFP and mCherry were investigated. Cultivations were performed applying special microtiter plates with optodes for dissolved oxygen tension (DOT) and pH measurement in the BioLector system. In this way, microbial growth, protein formation, DOT and pH were monitored on-line via optical signals. During these studies it became obvious that fluorescent proteins can interfere with the optical signals leading to incorrect results. In this work these effects are characterized in detail and possibilities are presented how such adverse effects can be corrected or minimized by mathematical procedures or modification of the measuring method. Additionally, it is shown that morphological changes of cells can affect the biomass on-line monitoring via scattered light. The here reported phenomena refer to typical experiments in biotechnological labs. For this reason these aspects are highlighted in this work to make operators of such valuable techniques as the BioLector aware for potential pitfalls and resulting misinterpretations. With the right approach it is possible to minimize existing problems and deal with them.
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影响因子：
7.4
作者：
Borisov, S. M.;Nuss, G.;Klimant, I.
通讯作者：
Klimant, I.
影响因子：
7.4
作者：
Kocincova, Anna S.;Borisov, Sergey M.;Wolfbeis, Otto S.
通讯作者：
Wolfbeis, Otto S.
影响因子：
3.8
作者：
Jeude, M.;Dittrich, B.;Buechs, J.
通讯作者：
Buechs, J.
影响因子：
4.9
作者：
Aigner, Daniel;Borisov, Sergey M.;Klimant, Ingo
通讯作者：
Klimant, Ingo
影响因子：
4.4
作者：
Drepper, Thomas;Huber, Robert;Jaeger, Karl-Erich
通讯作者：
Jaeger, Karl-Erich