Marshall Ryan, Noireaux Vincent
Physics and Nanotechnology, University of Minnesota, Minneapolis, MN, USA.
Methods Mol Biol. 2018;1772:61-93. doi: 10.1007/978-1-4939-7795-6_4.
Over the past decade, a new generation of cell-free transcription-translation (TXTL) systems has been devised for emerging multidisciplinary applications. The DNA-dependent in vitro protein synthesis technology has been developed to tackle applications in synthetic biology, biological and chemical engineering, as well as quantitative disciplines such as biophysics. In addition to being convenient at the biosafety level, the new TXTL platforms are user-friendly; more affordable; more versatile at the level of transcription, with a TX repertoire covering hundreds of parts; and more powerful, with protein production reaching a few mg/mL in batch and continuous modes. As a consequence, TXTL is rising up as a popular research tool and is used by a growing research community. While TXTL is proving reliable for an increasing number of applications, it is important to gain appropriate TXTL skills, especially for quantitative applications. TXTL has become particularly useful to rapidly prototype genetic devices , from single regulatory elements to elementary circuit motifs . In this chapter, we describe the basic procedures to develop appropriate TXTL practices for the characterization of such genetic parts. We use an all E. coli TXTL system developed in our lab, now commercialized by Arbor Biosciences under the name myTXTL.
在过去十年中，新一代无细胞转录-翻译（TXTL）系统被设计用于新兴的多学科应用。依赖DNA的体外蛋白质合成技术已得到发展，以应对合成生物学、生物与化学工程以及生物物理学等定量学科中的应用。除了在生物安全层面方便之外，新的TXTL平台还用户友好；价格更亲民；在转录水平上更具通用性，转录元件库涵盖数百个元件；功能更强大，在批次和连续模式下蛋白质产量可达几毫克/毫升。因此，TXTL正在崛起成为一种流行的研究工具，并被越来越多的研究群体所使用。虽然TXTL在越来越多的应用中被证明是可靠的，但掌握适当的TXTL技能很重要，尤其是对于定量应用。TXTL对于从单个调控元件到基本电路基序的基因装置快速原型制作变得特别有用。在本章中，我们描述了为表征此类基因元件开发适当TXTL实践的基本程序。我们使用在我们实验室开发的全大肠杆菌TXTL系统，该系统现已由Arbor Biosciences以myTXTL的名称商业化。
