Raytheon BBN Technologies, Cambridge, MA, United States of America.
Department of Life Sciences and IC-Centre for Synthetic Biology, Imperial College London, London, United Kingdom.
PLoS One. 2021 Jun 7;16(6):e0252263. doi: 10.1371/journal.pone.0252263. eCollection 2021.
Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.
可重复性是合成生物学的一个关键挑战，但可重复性的基础只有在建立在可靠的参考材料之上才是坚实的。在这里，我们关注的是用工程遗传构建体转化的细菌的荧光测量的可重复性。这项比较分析包括三个大型的使用流式细胞仪和微孔板读数器、相同的遗传构建体和兼容的单位校准协议的实验室间研究。在所有三项研究中，我们发现微孔板读数器使用的校准剂具有同样高的精度。我们还发现，荧光测量与流式细胞仪结果和两年的微孔板读数器结果非常吻合，平均标准偏差为 1.52 倍，而第三年的微孔板读数器结果则一直偏移一个数量级以上，平均偏移 28.9 倍。分析可能的误差源表明，这种偏移是由于荧光素校准剂的不正确制备造成的。这些发现表明，从工程构建体中测量荧光具有高度的可重复性，但也迫切需要获得质量控制良好的用于微孔板读数器的荧光校准剂。
