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Cell-free gene expression: an expanded repertoire of applications


Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits.

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Fig. 1: Timeline of cell-free gene expression systems.
Fig. 2: Cell-free gene expression for prototyping cellular mechanisms of transcription and translation.
Fig. 3: Cell-free systems for prototyping proteins and pathways.
Fig. 4: Building cells and synthetic biology education.
Fig. 5: Design of cell-free biosensors.
Fig. 6: Cell-free biomanufacturing platforms.
Fig. 7: The economics of cell-free gene expression.


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The authors would like to acknowledge members of the Jewett and Lucks Labs for helpful discussions. They also specifically thank R. Murray, J. Swartz, K. Pardee, P. Freemont, J. Collins, and J. Lucks for discussions and input. M.C.J. acknowledges support from the Army Research Office Grants W911NF-16-1-0372, W911NF-19-1-0298 and W911NF-18-1-0200; National Science Foundation Grant MCB-1716766, the Air Force Research Laboratory Center of Excellence Grant FA8650-15-2-5518, the Defense Threat Reduction Agency Grant HDTRA1-15-10052/P00001, the Department of Energy Grant DE-SC0018249, the DOE Joint Genome Institute, the DARPA 1000 Molecules Program HR0011-15-C-0084, the Office of Energy Efficiency and Renewable Energy Grant DE-EE0008343, the Human Frontiers Science Program Grant RGP0015/2017, the National Institutes of Health Grant 1U19AI142780-01, the David and Lucile Packard Foundation, and the Camille Dreyfus Teacher-Scholar Program. A.D.S. was supported in part by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University’s Biotechnology Training Program. The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of Air Force Research Laboratory, Air Force Office of Scientific Research, DARPA, Defense Threat Reduction Agency or the U.S. Government.

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Nature Reviews Genetics thanks E. Strychalski and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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The authors contributed to all aspects of the article.

Corresponding author

Correspondence to Michael C. Jewett.

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M.C.J. has a financial interest in SwiftScale Biologics and Design Pharmaceuticals Inc. M.C.J.’s interests are reviewed and managed by Northwestern University in accordance with their conflict of interest policies. All other authors declare no conflicts of interest.

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The modular assembly of electronic circuits by combining well-characterized components (for example, resistors, voltage sources) onto a plastic board; the term has been co-opted for synthetic biology to describe the assembly of genetic regulatory networks.

Logic gate

An electronic component that can compute a basic Boolean binary operation (for example, AND, OR, NOT).

Sigma factors

The accessory protein components that form the holo bacterial RNA polymerase capable of transcription initiation.


An effect where downstream effectors cause a genetic load on upstream components of a multicomponent metabolic or genetic network.

Ring oscillator

A genetic circuit in which three species are interchanged (A → B → C → A) and their concentrations vary with a fixed period.

Protospacer-adjacent motif

(PAM). A 3′-recognition sequence for CRISPR–Cas proteins that determines the sites of RNA-mediated DNA cleavage.


Spherical compartment composed of phospholipid bilayers that spontaneously forms when fatty acids aggregate in water.


Liquid–liquid phase separation that occurs in polymer solutions.

Giant unilamellar vesicles

Liposomes containing a single bilayer with a size in the order of a whole cell or larger (radius ~1–100 µm).

Molecular beacon

A nucleic acid duplex in which one strand is covalently linked to a fluorophore and the other is linked to a fluorescence quencher, so that fluorescence is only observed upon disruption of the duplex.

Toehold switch

A genetic regulator in which mRNA translation is inhibited by sterically blocking a ribosome-binding site with a complementary strand of mRNA in the 5′ untranslated region, which can only be translationally competent if the duplex is disrupted by a complementary (trigger) strand.


Molecules that are not produced in any known natural biological systems.


Small vesicles originating from the endoplasmic reticulum when cells are sheared and centrifuged.

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Silverman, A.D., Karim, A.S. & Jewett, M.C. Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet 21, 151–170 (2020).

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