Cell-Free Expression Systems: From Gene Circuits to Self-Assembly Processes in a Test Tube

Abstract

Cell-free transcription-translation (TX-TL) systems are becoming powerful platforms to construct complex biological systems in vitro through the expression of DNA programs. Considerable efforts have been made to improve those systems in the past decade. Our laboratory has developed the most efficient and versatile in vitro E. coli TX-TL system to express synthetic or natural DNA programs encoding for self-assembly processes. Either in test tube reactions, in emulsion droplets or in liposomes, cell-free TX-TL reactions are now used for research studies ranging from elementary gene circuits to minimal cells.I will present our custom-made cell-free TX-TL system, its current capabilities and limitations. Recently we have shown that entire phages can be synthesized in cell-free TX-TL reactions from their genomes. The bacteriophage T7, composed of about 60 genes, is entirely synthesized in a single test tube reaction from its 40 kbp genomic DNA. Replication of the T7 DNA instructions occurs concurrently with phage expression and self-assembly. The phage phiX174 composed of a dozen of genes can also be synthesized from its genome. Encapsulated inside cell-sized phospholipid liposomes, the TX-TL system is used to construct a prototype of minimal cell. I will present this cell-free synthetic biology platform, our last experiments and how this system can be used to study the relationship between information and self-organization.