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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. Computational Design of Self-Assembling Peptide Chassis Materials for Synthetic Cells

   https://doi.org/10.1039/D2ME00169A  

   Ma, Yutao; Kapoor, Rohan; Sharma, Bineet; Liu, Allen; Ferguson, Andrew L (January 2022, Molecular Systems Design & Engineering)

   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 an alternative chassis material for synthetic cells that are more robust and stable than lipid vesicles, and can withstand harsh conditions including pH, thermal, and osmotic variations. In this work, we combine coarse-grained molecular simulation, enhanced sampling free energy calculations, Gaussian process regression, and Bayesian optimization to construct an active learning screening for diblock amphiphilic elastin-like polypeptides capable of forming thermodynamically stable vesicular structures suitable for the self-assembly of synthetic peptide vesicles. Our computational screen identifies a number of promising sequences that form peptidic vesicles with high thermodynamic stabilities relative to isolated peptides in bulk solvent on the order of 10-15 k B T per amino acid residue. 

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

   https://doi.org/10.1039/D1CC05579H  

   Sharma, Bineet; Ma, Yutao; Hiraki, Harrison L; Baker, Brendon M; Ferguson, Andrew L; Liu, Allen P (December 2021, Chemical Communications)

   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. 

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5. Lipid sponge droplets as programmable synthetic organelles

   https://doi.org/10.1073/pnas.2004408117  

   Bhattacharya, Ahanjit; Niederholtmeyer, Henrike; Podolsky, Kira A.; Bhattacharya, Rupak; Song, Jing-Jin; Brea, Roberto J.; Tsai, Chu-Hsien; Sinha, Sunil K.; Devaraj, Neal K. (August 2020, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems. 

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