Adir Omer, Sharf-Pauker Noga, Chen Gal, Kaduri Maya, Krinsky Nitzan, Shainsky-Roitman Janna, Shklover Jeny, Schroeder Avi
Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology; The Norman Seiden Multidisciplinary Program for Nanoscience and Nanotechnology, Technion - Israel Institute of Technology.
Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology; The Interdisciplinary Program for Biotechnology, Technion - Israel Institute of Technology.
J Vis Exp. 2020 Apr 27;158(158):e60829. doi: 10.3791/60829.
The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA→RNA→protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.
用于构建合成细胞的自下而上组装方法是在细胞模拟环境中分离和研究细胞过程的有效工具。此外，无细胞表达系统的发展已证明能够以可控方式利用合成生物学来重构蛋白质生产、转录和翻译过程（DNA→RNA→蛋白质）。在此，我们描述了一种制备无细胞表达系统的方案，包括制备高效细菌裂解物并将该裂解物封装在富含胆固醇的基于脂质的巨型单层囊泡（GUVs）（即稳定脂质体）内以形成合成细胞。该方案描述了制备合成细胞各组分的方法，包括活性细菌裂解物的制备，随后基于油包水乳液转移法详细逐步制备合成细胞。这些方法有助于以简单且经济的方式生产数百万个合成细胞，并且在生产不同类型蛋白质方面具有高度通用性。所获得的合成细胞可用于在隔离环境中研究蛋白质/RNA的生产和活性、进行定向进化，还可作为可控药物递送平台，用于在体内按需生产治疗性蛋白质。
