Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation

Abstract

The construction of genetically encoded cellular mimics in compartments containing organized synthetic cytosols is desirable for the development of artificial cells. Phase separated aqueous domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aqueous two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aqueous two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aqueous three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liquid organelles.

All-Aqueous Electrosprayed Emulsion for Templated Fabrication of Cytocompatible Microcapsules

“…When the osmolarity of the emulsion phase is decreased by reducing the concentration of dextran from 15 wt% to 5 wt%, we 14 observe that the resultant droplets shrink due to the diffusion of water from the emulsion to the continuous phase. Taking advantage of such an osmotic effect 39,40 , the final volume of the particles can be adjusted by varying the osmolarity of the two aqueous phases, as shown in The all-aqueous electrospray represents a simple step for encapsulating bioactive proteins in hydrogel particles. As a model of bioactive protein, 1 wt% β-amylase is added into the emulsion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Since the AAE approach provides mild conditions for gelation of hydrogel particles, the cytocompatibility of this approach is further tested by encapsulating living cells.…”

In vitro gene expression within membrane-free coacervate protocells

“…2,3 As a consequence, advances in protocell engineering are contributing to a resurgence in the use and development of in vitro gene expression systems (IVGES), 4,5 which provide an attractive approach to off-line biological information storage and processing in synthetic cell-free environments. 12,13 Cell-free gene expression systems have also been encapsulated within water-in-oil emulsion droplets, 14 as well as in non-lipid membrane-bound protocells produced by the spontaneous assembly of protein-polymer nanoconjugates (proteinosomes) 15 or amphiphilic silica nanoparticles (colloidosomes), 16 and in molecularly crowded environments such as aqueous dextran/polyethylene glycol (PEG) two-phase water-inoil emulsion droplets 17 and condensed droplets of a cell lysate. [6][7][8] This approach has provided important steps towards the construction of protocells with archetypal properties such as RNA replication, 9 sequential gene expression, 10 membrane-pore (a-hemolysin) assembly, 7 gene switching behaviour, 11 and extra-vesicular signalling to bacterial cells.…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[18,70] Chemical modifications have recently been used to increaset he propensityo fp roteins to undergo LLPS through, for example, protein supercharging [71] or covalent attachment of ionic polypeptidetags. [77] These studies demonstrate that supramolecular ordering is sufficient to favouro ligonucleotide templatinga nd to enhance non-enzymatic oligonucleotide polymerisation.M ore complex, multistep reactions were also achieved in ATPS and coacervate droplets;t hey included cell-free transcription/translation [78] and concerted cascade enzyme reactions, [79] with reported enhanced reaction rates or yields. [73] How their localised up-concentration in a crowded phase affects their biochemical reactions is an area of current study.C o-localisation of partner reactants (e.g.,e nzymes and substrates) might accelerate biochemical reactions: [74] the accumulation of hammerhead ribozymes in the dextran-rich phase of aP EG/dextran ATPS, for instance, was reported to enhance RNA catalysis rates, due to ac ombination of localised RNA up-concentration and crowding effects.…”