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1. 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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2. 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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3. 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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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. Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

   https://doi.org/10.1038/s41467-021-22329-3  

   Hershewe, Jasmine M. ; Warfel, Katherine F. ; Iyer, Shaelyn M. ; Peruzzi, Justin A. ; Sullivan, Claretta J. ; Roth, Eric W. ; DeLisa, Matthew P. ; Kamat, Neha P. ; Jewett, Michael C. ( December 2021 , Nature Communications)

   null (Ed.)

   Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli- based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N- linked and O -linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities. 

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