Raytheon BBN Technologies, Cambridge, MA, United States of America.
iGEM Foundation, Cambridge, MA, United States of America.
PLoS One. 2018 Jun 21;13(6):e0199432. doi: 10.1371/journal.pone.0199432. eCollection 2018.
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.
荧光报告物通常用于定量分析天然和工程细胞的活性或特性。然而，荧光通常仅以任意或归一化单位报告，而不是使用独立的校准标准品定义单位，这对于科学再现性来说是有问题的，对于有效的工程设计来说更是如此。在本文中，我们报告了一项实验室间研究，表明简单、低成本的单位校准方案可以纠正这种情况，产生可比的单位，并大大提高任意单位和归一化单位的精度。来自世界各地 92 个机构的参与者使用专为平板读数器和/或流式细胞仪设计的简单、低成本的单位校准方案，测量了转化为三种工程测试质粒的大肠杆菌的荧光，以及阳性和阴性对照。除了提供可比的单位外，独立校准标准品的使用还允许对阳性和阴性对照进行定量使用，以识别可能存在的方案失败情况。因此，独立校准标准品的使用允许精度提高数量级，与归一化单位相比，我们的研究中测量的 95%置信区间缩小了 600 倍。
