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Artificial cell based on in vitro gene expression

Vincent Noireaux, Ph.D.

With our present understanding of cellular structure and function, the realization of an artificial cell seems conceivable. Based on our knowledge of molecular biology and soft condensed matter, a constructive approach can be devised, inspired by the automata theory of J. v. Neumann. With the advances in cell-free expression a cell-like structure can be assembled from an extract supporting transcription and translation. First, we used a wheat germ extract to reconstitute elementary networks in vitro (1). Then, an Escherichia coli extract has been used to make a vesicle bioreactor that can sustain cell free expression for a few days (2). We are trying to develop this system to build a minimal cell. We are especially interested in the molecular and supramolecular mechanisms to get a self-reproducing system as well as the aspects of origin of life at the mesoscopic level.


Expression of alpha-hemolysin-eGFP inside a vesicle (see ref (2)). (A) Kinetics of expression: closed circles: 0.5nM pIVEX2.3d-alpha-Hemolysin-eGFP. Inset: blow up of the first 10 hours of expression of alpha-hemolysin-eGFP. For comparison, time course of expression of eGFP inside a vesicle without alpha-hemolysin under low osmotic pressure (closed squares, curve from Fig. 2B, 0.5nM pIVEX2.3d-eGFP). (B) Expression of alpha-hemolysin fused to eGFP after a few tens of hours inside a single vesicle (top) and an aggregate of vesicles (bottom), The E. coli extract is encapsulated in the vesicles with the plasmid pIVEX2.3d-alpha-Hemolysin-eGFP (0.5nM) surrounded by a feeding solution. Scale bar 20µm.

References

(1) Vincent Noireaux, Roy Bar-Ziv, and Albert Libchaber (2003) Principles of cell-free genetic circuit assembly PNAS 100: 12672-12677
(2) Vincent Noireaux and Albert Libchaber (2004) A vesicle bioreactor as a step toward an artificial cell assembly PNAS 101: 17669-17674.