Cell-Free Systems for Synthetic Biology

Cell-free systems provide a valuable platform for understanding, using, and expanding the capabilities of natural systems. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. As a consequence, cell-free systems provide an unprecedented and otherwise unattainable freedom of design to modify and control biological systems. In this presentation, I will describe both top-down and bottom-up efforts to design, construct, and modify biological catalysts involved in protein synthesis. In one example, I will describe our efforts to establish the first crude extract based cell-free protein synthesis (CFPS) platform based on an Escherichia coli strain lacking release factor 1 (RF1) and capable of high-level batch expression (>1.5g/L) of pure proteins harboring non-standard amino acids (nsAAs). Our yields outperform the best expression of proteins with single or multiple nsAAs in vivo by >10x, which widens the aperture to new diverse applications in functional biomaterials and medically relevant proteins. In another example, I will describe a bottom-up approach for constructing minimal cells in vitro. Towards this objective, we show significant progress in building a synthetic ribosome, the most complex of the macromolecular catalysts needed to boot-up self-replication. This goal has been precluded for decades because conventional E. coli ribosome reconstitutions are non-physiological, and ribosomes reconstituted with in vitro transcribed ribosomal RNA (rRNA) are essentially non-functional. To move beyond previous limitations, we developed an integrated synthesis, assembly, and translation method (called iSAT) that enables efficient one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into E. coli ribosomes, and synthesis of functional protein by these ribosomes in a ribosome free S150 extract. A novel feature of iSAT is that it mimics co-transcription of rRNA and ribosome assembly as it occurs in vivo. We also developed a ribosome synthesis and evolution method (termed RISE) for the selection of variant ribosomes in vitro. We have used RISE, the first purely in vitro method for ribosome engineering, to evolve the large ribosomal subunit for altered functionalities. We anticipate that iSAT and RISE will aid studies of ribosome biogenesis, make possible minimal cells, and catalyze a new paradigm for the synthesis and evolution of abiological polymers. Taken together, our complementary approaches are enabling a deeper understanding of why nature’s designs work the way they do and opening new frontiers for harnessing a dramatically expanded genetic code for manufacturing novel therapeutics and synthesizing genetically-encoded materials.