Methods to reduce variability in E. Coli-based cell-free protein expression experiments
Methods to reduce variability in E. Coli-based cell-free protein expression experiments
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DOI：
10.1016/j.synbio.2019.10.003
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发表时间：
2019-12-01
影响因子：
4.8
通讯作者：
Reuel, Nigel F.
中科院分区：
文献类型：
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作者：
Dopp, Jared L.;Jo, Yeong Ran;Reuel, Nigel F.
无细胞蛋白质合成（CFPS）是一种成熟的生物技术工具，在许多应用中显示出巨大的实用性，如蛋白质原型，构建遗传电路，设计生物传感器和表达细胞毒性蛋白。虽然CFPS已被广泛部署，但文献中提出的许多不同方法对于新用户来说可能具有挑战性。根据我们的经验和其他新进入该领域的人的经验，将CFPS作为实验室应用的最令人沮丧的方面之一可能是实验重复中存在的大量变异性。在这里，我们提供了一个回顾性总结CFPS方法，减少变异显着。这些方法包括优化提取物制备、完全溶解主混合物组分以及仔细混合反应物。这使我们的变异系数从97.3%降低到1.2%。此外，这些方法允许完全新手（例如学期轮换的本科生）提供与有经验的用户相当的数据，从而允许更广泛地参与这一令人兴奋的研究领域。
Cell-free protein synthesis (CFPS) is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins, building genetic circuits, designing biosensors, and expressing cytotoxic proteins. Although CFPS has been widely deployed, the many, varied methods presented in the literature can be challenging for new users to adopt. From our experience and others who newly enter the field, one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental replicates. Herein we provide a retrospective summary of CFPS methods that reduce variability significantly. These methods include optimized extract preparation, fully solubilizing the master mix components, and careful mixing of the reaction. These have reduced our coefficient of variation from 97.3% to 1.2%. Moreover, these methods allow complete novices (e.g. semester rotation undergraduate students) to provide data that is comparable to experienced users, thus allowing broader participation in this exciting research area.