Synthetic Biology with a Cell-Free TX-TL System: Metabolism, Gene Circuits and Minimal Cell in a Test Tube

Cell-free transcription-translation (TX-TL) systems were developed in the 60s to study the process of protein synthesis and gene regulation in bacteria. With the invention of highly efficient cell-free expression systems in the 90s, in vitro protein synthesis became used for biotechnological applications and in the industry as an alternative to the recombinant protein technology. Over the past decade, cell-free TX-TL systems have become promising platforms for in vitro synthetic biology and bioengineering.

I will present a cell-free TX-TL toolbox specifically developed to construct in vitro biochemical systems programmed with DNA. Our original idea was to reconstruct elementary gene circuits in isolation free of endogenous genetic material. Recently we showed that our custom-made TX-TL system can execute large DNA programs. The bacteriophage T7, composed of about 60 genes, is entirely synthesized in a single test tube reaction from its genomic DNA. Replication of the T7 DNA instructions occurs concurrently with phage expression and self-assembly. Encapsulated inside cell-sized liposomes, the TX-TL system is used to construct a prototype of minimal cell. I will present this cell-free TX-TL platform, our last experiments and how this system is used to engineer gene circuits and self-organized entities as well as how we can address biophysical questions.