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

   https://doi.org/10.1039/d0sm01644f  

   Sharma, Bineet; Ma, Yutao; Ferguson, Andrew L.; Liu, Allen P. (December 2020, Soft Matter)

   null (Ed.)

   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. 

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2. Facile Formation of Giant Elastin-like Polypeptide Vesicles as Synthetic Cells

   https://doi.org/10.26434/chemrxiv.13718194.v1  

   Bineet Sharma, Yutao Ma (February 2021, ChemRxiv)

   null (Ed.)

   Creating a suitable compartment for synthetic cells has led the exploration of different cell chassis materials from phospholipids to polymer to protein-polymer conjugates. Currently, the majority of cell-like compartments are made of lipid molecules as the resulting membrane resembles that of a natural cell. However, cell-sized lipid vesicles are prone to physical and chemical stresses and can be unstable in hosting biochemical reactions within. Recently, peptide vesicles that are more robust and stable were developed as a new chassis material for synthetic cells. Here we demonstrate the facile and robust generation of giant peptide vesicles made of elastin-like polypeptides (ELPs) by using an emulsion transfer method. We show that these peptide vesicles can stably encapsulate molecules and can host cell-free expression reactions. We also demonstrate membrane incorporation of another amphiphilic ELP into existing peptide vesicles. Since ELPs are genetically encoded, the approaches presented here provide exciting opportunities to engineer synthetic cell membranes. 

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3. Light-triggered explosion of lipid vesicles

   https://doi.org/10.1039/D0SM01027H  

   Malik, Vinit Kumar; Shin, Sangwoo; Feng, Jie (October 2020, Soft Matter)

   null (Ed.)

   Lipid vesicles have received considerable interest because of their applications to in vitro reductionist cell membrane models as well as therapeutic delivery vehicles. In these contexts, the mechanical response of vesicles in nonequilibrium environments plays a key role in determining the corresponding dynamics. A common understanding of the response of lipid vesicles upon exposure to a hypotonic solution is a characteristic pulsatile behavior. Recent experiments, however, have shown vesicles exploding under an osmotic shock generated by photo-reactions, yet the explanatory mechanism is unknown. Here we present a generalized biophysical model incorporating a stochastic account of membrane rupture to describe both swell-burst-reseal cycling and exploding dynamics. This model agrees well with experimental observations, and it unravels that the sudden osmotic shock strains the vesicle at an extreme rate, driving the vesicle into buckling instabilities responsible for membrane fragmentation, i.e. explosion. Our work not only advances the fundamental framework for non-equilibrium vesicle dynamics under osmotic stress, but also offers design guidelines for programmable vesicle-encapsulated substance release in therapeutic carriers. 

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4. Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

   https://doi.org/10.3791/66595  

   Loi, Kyle J; Moghimianavval, Hossein; Liu, Allen P (March 2024, Journal of Visualized Experiments)

   Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While expression of soluble proteins is usually successful in common CFE systems, reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful co-translational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells. 

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5. Bioactive cell-like hybrids from dendrimersomes with a human cell membrane and its components

   https://doi.org/10.1073/pnas.1811307116  

   Yadavalli, Srujana S.; Xiao, Qi; Sherman, Samuel E.; Hasley, William D.; Klein, Michael L.; Goulian, Mark; Percec, Virgil (December 2018, Proceedings of the National Academy of Sciences)

   Cell-like hybrids from natural and synthetic amphiphiles provide a platform to engineer functions of synthetic cells and protocells. Cell membranes and vesicles prepared from human cell membranes are relatively unstable in vitro and therefore are difficult to study. The thicknesses of biological membranes and vesicles self-assembled from amphiphilic Janus dendrimers, known as dendrimersomes, are comparable. This feature facilitated the coassembly of functional cell-like hybrid vesicles from giant dendrimersomes and bacterial membrane vesicles generated from the very stable bacterialEscherichia colicell after enzymatic degradation of its outer membrane. Human cells are fragile and require only mild centrifugation to be dismantled and subsequently reconstituted into vesicles. Here we report the coassembly of human membrane vesicles with dendrimersomes. The resulting giant hybrid vesicles containing human cell membranes, their components, and Janus dendrimers are stable for at least 1 y. To demonstrate the utility of cell-like hybrid vesicles, hybrids from dendrimersomes and bacterial membrane vesicles containing YadA, a bacterial adhesin protein, were prepared. The latter cell-like hybrids were recognized by human cells, allowing for adhesion and entry of the hybrid bacterial vesicles into human cells in vitro. 

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