Synthetic Biology with a Cell-Free TX-TL System: Metabolism, Gene Circuits, Phages and Artificial Cell

Cell-free transcription-translation (TX-TL) systems are becoming valuable platform for synthetic biology. In vitro TX-TL is now used for many applications ranging from protein evolution to therapeutics and high-throughput production. Noireaux’s lab has been interested in using cell-free TX-TL to execute DNA programs in vitro.

I will present a unique cell-free TX-TL platform specifically engineered to construct biochemical systems in test tubes (Figure 1). Our research project has three parts. We develop new metabolism pathways to fuel cell-free TX-TL reactions. We showed that simple carbon sources are as efficient as the expensive phosphate donor-kinase systems to regenerate ATP. Not only protein synthesis is strong, the degradation of the synthesized proteins using the endogenous AAA+ proteases is also much more efficient. We construct synthetic gene circuits, such as regulatory networks, to make quantitative models of gene regulation. The goal is to use these circuit modules to coordinate the expression of complex cellular functions. Recently we demonstrated that our platform can execute large DNA programs that encodes complex self-assembled entities. The phage T7 composed of about 60 genes is entirely expressed and synthesized in the cell-free TX-TL reactions. The E. coli MreB cytoskeleton was reconstituted in liposomes. The ultimate goal of this research program is the synthesis of a minimal cell. This is done by integrating metabolism, information and self-assembly processes and unraveling the cooperative link between these three indispensable components. I will present the last experiments on those topics.