Garenne David, Thompson Seth, Brisson Amaury, Khakimzhan Aset, Noireaux Vincent
School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, USA.
Synth Biol (Oxf). 2021 Aug 4;6(1):ysab017. doi: 10.1093/synbio/ysab017. eCollection 2021.
The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems over a remarkable scope of applications and physical scales. As the utilization of DNA-directed protein synthesis expands in scope, developing more powerful cell-free transcription-translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all- TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 10 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells.
新一代无细胞基因表达系统能够在广泛的应用和物理尺度范围内对生物系统进行原型设计和工程改造。随着DNA指导的蛋白质合成应用范围的扩大，开发更强大的无细胞转录-翻译（TXTL）平台仍然是执行更大DNA程序或提高无细胞生物制造能力的主要目标。在这项工作中，我们报告了全TXTL工具箱3.0的能力，这是一种专门为合成生物学开发的多功能无细胞表达系统。在非补料分批模式反应中，荧光报告蛋白增强型绿色荧光蛋白（eGFP）的合成量达到4毫克/毫升。在由装载TXTL反应的脂质体组成的合成细胞中，当为反应提供化学构建模块的物质通过膜通道扩散以促进与外部溶液的交换时，eGFP的产生浓度>8毫克/毫升。由40 kb基因组和约60个基因编码的噬菌体T7，其产生浓度为10个噬菌斑形成单位/毫升（PFU/ml）。该TXTL系统通过提供独特的优势和特性，扩展了当前的无细胞表达能力，可用于测试调控元件和回路、生物制造生物制品或构建合成细胞。
