Characterizing and prototyping genetic networks with cell-free transcription–translation reactions

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative 'design–build–test' cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription–translation (TX–TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX–TL system that utilizes the native Escherichia coli TX–TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX–TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX–TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems.