The cell-free molecular synthesis of biochemical systems is a rapidly growing field of research. Advances in the Human Genome Project, DNA synthesis, and other technologies have allowed the in vitro construction of biochemical systems, termed cell-free biology, to emerge as an exciting domain of bioengineering. Cell-free biology ranges from the molecular to the cell-population scales, using an ever-expanding variety of experimental platforms and toolboxes. In this review, we discuss the ongoing efforts undertaken in the three major classes of cell-free biology methodologies, namely protein-based, nucleic acids–based, and cell-free transcription–translation systems, and provide our perspectives on the current challenges as well as the major goals in each of the subfields.

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