Triggered Gene Expression in Fed‐Vesicle Microreactors with a Multifunctional Membrane

Nourian, Zohreh; Roelofsen, Wouter; Danelon, Christophe

doi:10.1002/ange.201107123

Cited by 44 publications

(51 citation statements)

References 34 publications

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“…Next we sought to exploit the Spinach technology to visualize mRNA in protein‐synthesizing liposomes. The PURE system enzymes, cofactors and YFP–Spinach DNA template were compartmentalized inside surface‐tethered vesicles as previously described 1f. Gene expression was triggered by the external supply of the feeding solution, which contained the amino acids, nucleotide triphosphates and tRNAs along with 20 μ M DFHBI.…”

Section: Resultsmentioning

confidence: 99%

Unbiased Tracking of the Progression of mRNA and Protein Synthesis in Bulk and in Liposome‐Confined Reactions

et al. 2013

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The compartmentalization of a cell-free gene expression system inside a self-assembled lipid vesicle is envisioned as the simplest chassis for the construction of a minimal cell. Although crucial for its realization, quantitative understanding of the dynamics of gene expression in bulk and liposome-confined reactions is scarce. Here, we used two orthogonal fluorescence labeling tools to report the amounts of mRNA and protein produced in a reconstituted biosynthesis system, simultaneously and in real-time. The Spinach RNA aptamer and its fluorogenic probe were used for mRNA detection. Applying this dual-reporter assay to the analysis of transcript and protein production inside lipid vesicles revealed that their levels are uncorrelated, most probably a consequence of the low copy-number of some components in liposome-confined reactions. We believe that the stochastic nature of gene expression should be appreciated as a design principle for the assembly of a minimal cell.

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Section: Resultsmentioning

confidence: 99%

Unbiased Tracking of the Progression of mRNA and Protein Synthesis in Bulk and in Liposome‐Confined Reactions

et al. 2013

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“…This property has been exploited to enable feeding of external NTPs to encapsulated enzymatic reactions (125), although this method may require thermocycling between the membrane phase transition temperature and the temperature optimum of the enzyme (189). Alternatively, protein pores (133,190), shorterchain phospholipids (191), or detergents (192) can be used to permeabilize the membrane. Another attractive approach is to use vesicle fusion to deliver nutrients encapsulated in feeder vesicles (161); unfortunately, most simple vesicle fusion methods are inefficient and induce considerable contents leakage (158)(159)(160)193).…”

Section: Simple Protocellsmentioning

confidence: 99%

Progress Toward Synthetic Cells

Blain

Szostak²

2014

Annu. Rev. Biochem.

275

249

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The complexity of even the simplest known life forms makes efforts to synthesize living cells from inanimate components seem like a daunting task. However, recent progress toward the creation of synthetic cells, ranging from simple protocells to artificial cells approaching the complexity of bacteria, suggests that the synthesis of life is now a realistic goal. Protocell research, fueled by advances in the biophysics of primitive membranes and the chemistry of nucleic acid replication, is providing new insights into the origin of cellular life. Parallel efforts to construct more complex artificial cells, incorporating translational machinery and protein enzymes, are providing information about the requirements for protein-based life. We discuss recent advances and remaining challenges in the synthesis of artificial cells, the possibility of creating new forms of life distinct from existing biology, and the promise of this research for gaining a deeper understanding of the nature of living systems.

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“…More recently the rapid expansion of synthetic biology (11) has given additional conceptual stimuli and technical tools to this field, especially by the so-called bottom-up approach (12). Despite the recent progress, which is mainly focused on the reconstitution of essential biochemical functions inside confined environments (13) such as phospholipid (4,5,(14)(15)(16)(17)(18)(19) and fatty acid vesicles (8,20,21), water-in-oil (w/o) droplets (22), and coacervates (23), the primary generation of chemical energy by molecular machineries remains a missing key function.…”

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confidence: 99%

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells

Altamura

Milano

Tangorra

et al. 2017

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

107

105

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Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

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Triggered Gene Expression in Fed‐Vesicle Microreactors with a Multifunctional Membrane

Cited by 44 publications

References 34 publications

Unbiased Tracking of the Progression of mRNA and Protein Synthesis in Bulk and in Liposome‐Confined Reactions

Unbiased Tracking of the Progression of mRNA and Protein Synthesis in Bulk and in Liposome‐Confined Reactions

Progress Toward Synthetic Cells

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells

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