Internalization and observation of fluorescent biomolecules in living microorganisms via electroporation.
Internalization and observation of fluorescent biomolecules in living microorganisms via electroporation.
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DOI：
10.3791/52208
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发表时间：
2015-02-08
期刊：
影响因子：
--
通讯作者：
Kapanidis AN
中科院分区：
文献类型：
--
作者：
Aigrain L;Sustarsic M;Crawford R;Plochowietz A;Kapanidis AN
研究生物分子在体内的能力对于理解它们在生物学背景下的功能是至关重要的。一种有效的方法是将荧光蛋白（如GFP）感兴趣的分子融合在一起，研究它们的表达、定位和功能。然而，与通常用于体外实验的有机荧光团相比，绿色荧光蛋白及其衍生物明显更大，光稳定性更差，这限制了研究范围。我们最近介绍了一种基于电穿孔的简单、通用和高通量的方法，允许用有机荧光团标记的生物分子内在化到活的微生物中。在这里，我们描述了如何使用电穿孔将标记的DNA片段或蛋白质内化到大肠杆菌和酿酒酵母中，如何使用荧光显微镜量化内化分子的数量，以及如何量化电穿孔细胞的活力。数据可以获得在单细胞或单分子水平使用荧光或FRET。还提出了内化非标记分子触发体内可观察到的生理反应的可能性。最后，讨论了针对特定生物系统的协议优化策略。
The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.
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影响因子：
48
作者：
Dempsey, Graham T.;Vaughan, Joshua C.;Chen, Kok Hao;Bates, Mark;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1126/science.1247651
发表时间：
2014-02-14
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Jaitin DA;Kenigsberg E;Keren-Shaul H;Elefant N;Paul F;Zaretsky I;Mildner A;Cohen N;Jung S;Tanay A;Amit I
通讯作者：
Amit I
影响因子：
1.2
作者：
Uphoff, Stephan;Sherratt, David J.;Kapanidis, Achillefs N.
通讯作者：
Kapanidis, Achillefs N.
影响因子：
48
作者：
Sakon, John J.;Weninger, Keith R.
通讯作者：
Weninger, Keith R.
影响因子：
48
作者：
Tokunaga, Makio;Imamoto, Naoko;Sakata-Sogawa, Kumiko
通讯作者：
Sakata-Sogawa, Kumiko