Next Generation Cell-Free Expression Systems for Synthetic Biology

A new generation of cell-free expression systems, optimized for synthetic biology, has recently emerged. In vitro transcription-translation (TX-TL) is becoming a highly flexible and user-friendly technology for bioengineering. Cell-free TX-TL is rapidly expanding its scope of applications to include biological network prototyping, artificial cell systems and biological physics, nanotechnology, metabolic and chemical engineering, medicine, and the production of functional membrane proteins. In vitro protein synthesis is increasingly employed as a means to construct, understand and interrogate complex biochemical systems, ranging from molecular to cellular size, as well as a tool to expand the capabilities of natural systems.

In this presentation, I will first give an overview of cell-free TX-TL systems, their history and the recent emergence of a new generation of platforms. I will describe the capabilities of the current cell-free expression systems and their applications.

In the second part of the talk, I will describe the cell-free TX-TL system that my lab has recently developed along with its unique features. The repertoire of transcription covers all E. coli regulatory elements, far more than the conventional hybrid bacteriophage systems. We implemented novel metabolisms to stimulate protein synthesis up to 2 mg/ml in batch mode reactions and 6 mg/ml in semi-continuous mode. In cell-sized liposomes, continuous mode is achieved using the toxin alpha-hemolysin. This cell-free toolbox also integrates robust methods to tune the synthesis and degradation rates of messengers and proteins, thus facilitating the construction of dynamical systems in test tube reactions, microfluidics and synthetic cells. I will describe how these improvements can be exploited in new applications, including biosynthesis and biophysics.