Cell-Free Synthesis and Assembly of Protein Nano-Machines on a Chip

Towards the development of synthetic bio-devices that mimic basic cellular processes, we are using a novel photolithographic approach to carry out controlled gene expression reactions on a silicon chip [1]. Our approach uses surface-immobilized, highly dense gene compartments, reaching in vivo DNA densities, and localized antibodies that serve as traps for gene products [2, 3]. Addition of a cell-free extract results in a complete transcription-translation reaction that can be modulated by the orientation and density of immobilized DNA [4]. Protein products are localized on the antibody traps next to their gene source forming membrane-free compartments.

The cell-free biochip approach provides conceptual advantages for studying bio-assembly processes of large macromolecular complexes such as T4 bacteriophage and the ribosome: (i) Cell-free expression renders coupling between synthesis and assembly, better mimicking assembly in vivo (ii) Surface anchoring generates structural scaffolds for assembly [5]. We have already shown that expression of bacteriophage T4 tail protein resulted in correct self-assembly into long nanotubes on surface-patterned antibody traps, as imaged directly by transmission electron microscopy [2]. We are currently investigating the outcome of expanding the number of T4 genes that can be added to cell-free on-chip reactions and that would result in correct assembly. In addition we are developing tools to decipher viral and ribosome assembly pathways and explore the possibility to affect assembly outcome by the initial patterns of localized genes and antibody traps.

[1] Small

[2]Nature Nanotech 7, 374-378, 2012

[3] MSB 4, 181, 2008

[4]PNAS 107, (2010) 2836-2841

[5]WIREs Nanomed Nanobiotechnol, 5, (2013) 613–628