Quantification of bacterial fluorescence using independent calibrants.
Quantification of bacterial fluorescence using independent calibrants.
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DOI：
10.1371/journal.pone.0199432
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
with the iGEM Interlab Study Contributors
中科院分区：
文献类型：
--
作者：
Beal J;Haddock-Angelli T;Baldwin G;Gershater M;Dwijayanti A;Storch M;de Mora K;Lizarazo M;Rettberg R;with the iGEM Interlab Study Contributors
荧光记者通常用来量化天然细胞和工程细胞的活性或性质。然而，荧光仍然通常只以任意或归一化的单位来报告，而不是以使用独立校准剂定义的单位来报告，这对科学的重复性是有问题的，在涉及到有效的工程时更是如此。在这篇文章中，我们报告了一项实验室间的研究，表明简单、低成本的单位校准方案可以弥补这种情况，产生可比较的单位，并显著提高精度，无论是任意单位还是归一化单位。来自世界各地的92个机构的参与者使用简单、低成本的单位校准方案，测量了用三种工程测试质粒转化的大肠杆菌以及阳性和阴性对照的荧光，这些单位校准方案是为平板阅读器和/或流式细胞仪设计的。除了提供可比较的单位外，使用独立的校准剂还允许定量使用阳性和阴性对照，以确定协议故障的可能情况。因此，独立校准剂的使用可以使精度提高数量级，将我们研究中测量的95%可信区间缩小到归一化单位的600倍。
Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.
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