DNA-based communication in populations of synthetic protocells

Joesaar, Alex; Yang, Shuo; Bögels, Bas W.A.; Linden, Ardjan J. van der; Pieters, Pascal A.; Kumar, B. V. V. S. Pavan; Dalchau, Neil; Phillips, Andrew; Mann, Stephen; Greef, Tom F. A. de

doi:10.1038/s41565-019-0399-9

Cited by 266 publications

(347 citation statements)

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“…We were able to disassemble the target droplet without affecting the neighbouring droplets such that TAMRA-ssDNAw as ejected locally into the supernatant and subsequently captured by the adjacent non-fluorescent droplet, which showed ag radual increase in red fluorescence over ap eriod of approximately 100 s ( Figure 3f-h and Supporting Information, Movie S3). Fore xample,a sD NA has been used to achieve cell-free protein expression in coacervate droplets [14] and develop programmable logic gates in synthetic protocells, [42] lightactuated DNAc oacervation could be exploited to activate signalling pathways in populations of active droplets or synthetic protocells,p aving the way to the construction of life-like colloidal systems capable of spatiotemporally coordinated behaviours. Asimilar light-mediated pathway for the diffusion-limited trafficking of ssDNAw as demonstrated between multiple co-located coacervate microdroplets by sequential UV processing of individual TAMRA-ssDNAcontaining droplets and neighbouring unloaded droplets (Supporting Information, Figure S12).…”

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confidence: 99%

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

et al. 2019

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Coacervate microdroplets produced by liquid-liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane-free organelles in living systems.A chieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light-responsive coacervate droplets prepared from mixtures of double-stranded DNAa nd an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively,d ue to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides followt he dynamics of phase separation such that light-activated transfer,m ixing,h ybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets.O ur results open perspectives for the spatiotemporal control of DNAc oacervates and provide as tep towards the dynamic regulation of synthetic protocells.The dynamic compartmentalization of biological components in space and time is ah allmark of functional synchronization in living cells.Biomolecular condensates formed via liquid-liquid phase separation are dynamic subcellular compartments that are actively formed and dissolved in response to environmental cues. [1,2] They play acrucial role for example in the compaction of polynucleotides and regulation of genetic expression. [3,4] Microdroplets produced in vitro by associative (coacervates) or segregative (aqueous two-phase systems) liquid-liquid phase separation have recently been used as synthetic models of dynamic protocells. [5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression. [14] Polynucleotides have been used as scaffold components to assemble coacervate droplets, [15][16][17][18][19] and selective sequestration of guest polynucleotides has been demonstrated in preformed coacervates. [12,20,21] Owing to their liquid-like nature and the weak molecular interactions,s ynthetic coacervate droplets can dynamically respond to external stimuli. [7,22,23] Fori nstance,t heir formation and dissolution has been achieved by changes in pH, [5] temperature, [24][25][26] ionic strength, [27,28] and more recently by enzymemediated catalytic activity. [15,[29][30][31][32] However,p latforms enabling the rapid and localized actuation of polynucleotide microphase separation have not yet been developed. Due to its favourable spectral and spatiotemporal capabilities,l ight holds great promise as aprogrammable trigger for controlling the reversible assembly and disassembly of coacervate droplets.L ight has been used to control synthetic microcompartments, [33][34][35] trigger biomolecular condensation in cellulo via optogenetic tools, [36,37] promote the dynamical shaping of soft colloids, [38] and induce the reversible compaction of single DNAc hains. [39][40][41] Here,w ee xploit t...

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Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

et al. 2019

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“…Scientists have dedicated to the construction of synthetic natural cells and synthetic cell–cell communication systems . de Greef and co‐workers reported the DNA‐based communication between neighboring artificial cells . Most cell–cell signaling relies on the transport of signals through the cell membrane.…”

Section: Applications Of Fabricated Artificial Cellsmentioning

confidence: 99%

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

Xie

Xiong

et al. 2019

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Fabrication of artificial biomimetic materials has attracted abundant attention. As one of the subcategories of biomimetic materials, artificial cells are highly significant for multiple disciplines and their synthesis has been intensively pursued. In order to manufacture robust “alive” artificial cells with high throughput, easy operation, and precise control, flexible microfluidic techniques are widely utilized. Herein, recent advances in microfluidic‐based methods for the synthesis of droplets, vesicles, and artificial cells are summarized. First, the advances of droplet fabrication and manipulation on the T‐junction, flow‐focusing, and coflowing microfluidic devices are discussed. Then, the formation of unicompartmental and multicompartmental vesicles based on microfluidics are summarized. Furthermore, the engineering of droplet‐based and vesicle‐based artificial cells by microfluidics is also reviewed. Moreover, the artificial cells applied for imitating cell behavior and acting as bioreactors for synthetic biology are highlighted. Finally, the current challenges and future trends in microfluidic‐based artificial cells are discussed. This review should be helpful for researchers in the fields of microfluidics, biomaterial fabrication, and synthetic biology.

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“…Reprinted with permission from Springer Nature: Nature Nanotechnology , DNA‐based communication in populations of synthetic protocells. 14, 369–378, https://doi.org/10.1038/s41565‐019‐0399‐9 (Joesaar et al, 2019), Copyright (2019). CFPS, cell‐free protein synthesis [Color figure can be viewed at wileyonlinelibrary.com]…”

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

confidence: 99%

“…In this design, DNA strand‐displacement circuits were encapsulated inside streptavidin‐containing proteinosome by covalently crosslinked BSA–NH 2 /PNIPAAm (w/o) emulsion nanodroplets. Proteinosome further allowed for sensing, processing and responding to DNA‐based messages, cascaded amplification, bidirectional broadcasting and disseminated computational operations (Joesaar et al, 2019).…”

Section: Cfps: Latest Platforms and Applications In Biotechnology Andmentioning

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

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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.

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DNA-based communication in populations of synthetic protocells

Cited by 266 publications

References 56 publications

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

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

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