Quantification of Interlaboratory Cell-Free Protein Synthesis Variability
Quantification of Interlaboratory Cell-Free Protein Synthesis Variability
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DOI：
10.1021/acssynbio.9b00178
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发表时间：
2019-09-01
影响因子：
4.7
通讯作者：
Lux, Matthew W.
中科院分区：
文献类型：
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作者：
Cole, Stephanie D.;Beabout, Kathryn;Lux, Matthew W.
无细胞蛋白质合成(CFPS)平台曾经主要是生产难以表达的蛋白质的研究工具，现在越来越多地被合成生物界用于生物制造、快速筛选系统和现场就绪传感器等应用。虽然个别研究的一致性在文献中是显而易见的，但在实验室之间，甚至在实验室内的个人之间，对复制结果的挑战被从业者公开讨论。随着该领域的不断发展和应用，对CFPS的预期变异性和潜在来源的相对贡献的定量了解将变得越来越重要。在这里，我们提供了第一个定量评估实验室间的可变性的CFPS。三个实验室实施了单一的CFPS方案，并进行了一系列的材料和人员交流，旨在量化与部位、操作员、细胞提取液制备和补充试剂制备相关的变异性的相对贡献。我们发现，在每个实验室准备的材料，成对交换，并在每个地点进行测试，结果是40.3%的变异系数，而单一操作员使用一套材料在几天内的变异系数为7.64%。试剂制剂对观察到的变异有很大贡献；然而，令人惊讶的是，提取物制剂没有解释任何观察到的变异，即使是在不同的实验室由不同的操作员制备的。随后的交流表明，场地和操作员各自都对观察到的实验室间变异性做出了贡献。除了首次对CFPS的实验室间可变性进行定量评估外，这些结果还为个体操作员在几天内的可变性建立了基线，可用作社区驱动的标准化工作的初始基准。我们预计，我们的结果将缩小未来的研究途径，以开发最佳实践，最终降低实验室间的变异性，加快研究进展，并告知CFPS适用于现实世界的应用。
Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.