Calcium-triggered DNA-mediated membrane fusion in synthetic cells.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

Calcium-triggered DNA-mediated membrane fusion in synthetic cells.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions.

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane-membrane interactions and possible future research directions.

Facile formation of giant elastin-like polypeptide vesicles as synthetic cells.

We demonstrate the facile and robust generation of giant peptide vesicles by using an emulsion transfer method. These robust vesicles can sustain chemical and physical stresses. The peptide vesicles can host cell-free expression reactions by encapsulating essential ingredients. We show the incorporation of another cell-free expressed elastin-like polypeptide into the existing membrane of the peptide vesicles.

In search of a novel chassis material for synthetic cells: emergence of synthetic peptide compartment.

Giant lipid vesicles have been used extensively as a synthetic cell model to recapitulate various life-like processes, including in vitro protein synthesis, DNA replication, and cytoskeleton organization. Cell-sized lipid vesicles are mechanically fragile in nature and prone to rupture due to osmotic stress, which limits their usability. Recently, peptide vesicles have been introduced as a synthetic cell model that would potentially overcome the aforementioned limitations. Peptide vesicles are robust, reasonably more stable than lipid vesicles and can withstand harsh conditions including pH, thermal, and osmotic variations. This mini-review summarizes the current state-of-the-art in the design, engineering, and realization of peptide-based chassis materials, including both experimental and computational work. We present an outlook for simulation-aided and data-driven design and experimental realization of engineered and multifunctional synthetic cells.

A bulk sub-femtoliter in vitro compartmentalization system using super-fine electrosprays.

The extreme miniaturization of biological and chemical assays in aqueous-droplet compartments enables spatiotemporal control for large-scale parallel experimentation and can thus permit new capabilities for "digitizing" directed molecular evolution methodologies. We report a remarkably facile bulk method to generate mega-scale monodisperse sub-femtoliter aqueous droplets by electrospray, using a prototype head with super-fine inkjet technology. Moreover, the electrostatic inkjet nozzle that injects the aqueous phase when immersed within an immiscible phase (an optimized oil/surfactant mixture) has the advantage of generating cell-like sub-femtoliter compartments for biomolecule encapsulation and successive biological and chemical reactions. Sub-femtoliter droplets of both liquid (water-in-oil, volumes ranging from 0.2 to 6.4 fL) and gel bead (agarose-in-oil, volume ranging from 0.3 to 15.6 fL) compartments with average sizes of 1.3 µm and 1.5 µm, respectively, were successfully generated using an inkjet nozzle at a speed of more than 10(5) droplets per second. We demonstrated the applicability of this system by synthesizing fluorescent proteins using a cell-free expression system inside electrosprayed sub-femtoliter droplets at an accelerated rate, thereby extending the utility of in vitro compartmentalization with improved analytical performance for a top-down artificial cellular system.