TXTL-based approach to synthetic cells

Garamella, Jonathan; Garenne, David; Noireaux, Vincent

doi:10.1016/bs.mie.2018.12.015

Cited by 22 publications

(29 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All of the performed reactions contained the same ingredients, except for the plasmids that were changed based on the proteins synthesized. The minimal cell system was prepared by encapsulating TXTL reactions into cell-sized liposomes, based on the emulsion transfer method (9, 31), following a procedure reported thoroughly by our group recently (32). The difference in osmolarity between the TXTL reactions encapsulated inside the liposomes and the external solution was measured to be between 25 and 75 mOsm, a magnitude that we found adequate for our study.…”

Section: Resultsmentioning

confidence: 99%

“…Lipid Membrane Composition and Molecular Crowding. We chose PC (egg phosphatidylcholine) as the default phospholipid, given that liposome production is the greatest with this lipid and with respect to the method employed in this work (32). Making hundreds of vesicles per preparation is practical in a context of deformation measurement.…”

Section: Resultsmentioning

confidence: 99%

“…Liposome Preparation. The protocol to prepare the TXTL-loaded liposomes, based on the emulsion method, has been described, in detail, by our group in a recent publication (32). Briefly, the phospholipids were dissolved in chloroform at a concentration of around 20 mg/mL.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods

Garenne

Libchaber

Noireaux

2020

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Executing gene circuits by cell-free transcription−translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a central limitation to the development of cell analogs genetically programmed. In this work, we express the Escherichia coli gene mreB inside vesicles with bilayers made of lipid-polyethylene glycol (PEG). We demonstrate that two-dimensional molecular crowding, emulated by the PEG molecules at the lipid bilayer, is enough to promote the polymerization of the protein MreB at the inner membrane into a sturdy cytoskeleton capable of transforming spherical liposomes into elongated shapes, such as rod-like compartments. We quantitatively describe this mechanism with respect to the size of liposomes, lipid composition of the membrane, crowding at the membrane, and strength of MreB synthesis. So far unexplored, molecular crowding at the surface of synthetic cells emerges as an additional development with potential broad applications. The symmetry breaking observed could be an important step toward compartment self-reproduction.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods

Garenne

Libchaber

Noireaux

2020

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…An indispensable tool for studies of synthetic biology, the origin of life, gene therapy, drug delivery, and so forth, is the synthetic cells, also referred to as artificial cells or protocells (Stano, 2019). Synthetic cells can afford compartments for the realization of CFPS, in the same way, TX‐TL machinery embedded inside synthetic cells can be used to build genetic circuits to mimic novel functions resembling living cells (Garamella, Garenne, & Noireaux, 2019). That is, synthetic biology traditionally utilizes genetic circuits to display complex spatiotemporal as well as collective behavior by establishing gene networks capable of establishing oscillations, synchronization, and combinatorial control of endogenous signaling pathways (Dubuc et al, 2019).…”

Section: Cfps: From Test Tube Reactions To Cell‐free Expression In MImentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

View full text Add to dashboard Cite

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

show abstract

“…Among them, liposome-based artificial cells (LBACs) have been one of the most commonly studied artificial cells. An LBAC consists of a liposome (small capsule made from a lipid bilayer membrane), a cell-free protein expression system (CFPES), and DNA(s) encoding proteins of interest [1]. They express proteins which perform the designated functions.…”

Section: Introductionmentioning

confidence: 99%

Construction of an Artificial Cell Capable of Protein Expression at Low Temperatures Using a Cell Extract Derived from Pseudomonas fluorescens

Fukumoto

Tonooka

2021

Processes

View full text Add to dashboard Cite

A liposome-based artificial cell (LBAC) consists of a liposome encapsulating a cell-free protein expression system (CFPES) and protein-encoding DNA. It is surrounded by a lipid bilayer membrane and synthesizes proteins that resemble actual cells. Hence, they have been one of the most studied artificial cells. According to recent studies, they have been able to sense bio-functional molecules by synthesizing fluorescent proteins in response to target molecules. Therefore, they are expected to be used as biosensors. However, previously reported LBACs encapsulated the CFPES derived from Escherichia coli, resulting in the most productive protein expression at 20–40 °C. To broaden the range of their working temperatures to lower temperatures, in this study, we constructed LBACs using a CFPES derived from Pseudomonas fluorescens that grows at a temperature range of 4 °C to 30 °C. We then demonstrated that the constructed LBAC expressed proteins at 8 °C and that, the protein expression capability of the LBAC derived from P. fluorescens was four-fold higher than that derived from E. coli at 8 °C. This study will pave the way for the development of artificial cell-based biosensors that work in cold environments or for the synthesis of heat-labile proteins in LBACs.

show abstract

TXTL-based approach to synthetic cells

Cited by 22 publications

References 31 publications

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Construction of an Artificial Cell Capable of Protein Expression at Low Temperatures Using a Cell Extract Derived from Pseudomonas fluorescens

Contact Info

Product

Resources

About