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

Soft Matter ◽  
2020 ◽  
Vol 16 (48) ◽  
pp. 10769-10780
Author(s):  
Bineet Sharma ◽  
Yutao Ma ◽  
Andrew L. Ferguson ◽  
Allen P. Liu
Keyword(s):  
Synthetic Peptide ◽  
Cell Model ◽  
Lipid Vesicles ◽  
Synthetic Cell ◽  
Synthetic Cells

Giant lipid vesicles have been used extensively as a synthetic cell model to recapitulate various life-like processes. In recent years, peptide vesicles are gaining attention as an alternative chassis material.

scholarly journals Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions

Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 912
Author(s):  
Bineet Sharma ◽  
Hossein Moghimianavval ◽  
Sung-Won Hwang ◽  
Allen P. Liu
Keyword(s):  
Membrane Fusion ◽  
Intercellular Communication ◽  
Lipid Vesicles ◽  
Bilayer Membrane ◽  
Future Research ◽  
Membrane Interactions ◽  
Protein Insertion ◽  
Synthetic Cell ◽  
Challenges And Opportunities ◽  
Synthetic Cells

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.

scholarly journals Facile Formation of Giant Elastin-like Polypeptide Vesicles as Synthetic Cells

2021 ◽  
Author(s):  
Bineet Sharma ◽  
Yutao Ma ◽  
Andrew Ferguson ◽  
Allen Liu
Keyword(s):  
Host Cell ◽  
Lipid Vesicles ◽  
Free Expression ◽  
Polymer Conjugates ◽  
Protein Polymer ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Transfer Method ◽  
Physical And Chemical ◽  
Natural Cell

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.

scholarly journals Decision-making in a synthetic cell: the limits of biological computation

2020 ◽  
Author(s):  
Ferdinand Greiss ◽  
Shirley S. Daube ◽  
Vincent Noireaux ◽  
Roy Bar-Ziv
Keyword(s):  
Decision Making ◽  
Single Molecule ◽  
Regulatory Networks ◽  
Cell Model ◽  
Energetic Cost ◽  
Single Molecule Level ◽  
Model Free ◽  
Gene Copies ◽  
Synthetic Cell ◽  
Synthetic Cells

AbstractWe measured the dynamics of decision-making by a minimal bistable gene network integrated in a synthetic cell model, free of external perturbations. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow computation to a fuzzy and rapid regime dominated by singleprotein fluctuations. Fuzzy computation appeared at DNA and protein concentrations 100-fold lower than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. Whereas the high-copy regime was characterized by a sharp transition, hysteresis and robust memory, the low-copy limit showed incipient strong fluctuations, switching between states, and a signature of cellular individuality across the decision-making point. Our work establishes synthetic cells operating rapidly at the single molecule level to integrate gene regulatory networks with metabolic pathways for sustained survival with low energetic cost.One Sentence SummaryDecision-making in a synthetic cell can be slow and precise or rapid and probabilistic by reducing the number of computing molecules by five decades down to single-molecule fluctuations.

scholarly journals Facile Formation of Giant Elastin-like Polypeptide Vesicles as Synthetic Cells

2021 ◽  
Author(s):  
Bineet Sharma ◽  
Yutao Ma ◽  
Andrew Ferguson ◽  
Allen Liu
Keyword(s):  
Host Cell ◽  
Lipid Vesicles ◽  
Free Expression ◽  
Polymer Conjugates ◽  
Protein Polymer ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Transfer Method ◽  
Physical And Chemical ◽  
Natural Cell

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.

scholarly journals Actuation of synthetic cells with proton gradients generated by light-harvesting E. coli

2020 ◽  
Author(s):  
Kevin Jahnke ◽  
Noah Ritzmann ◽  
Julius Fichtler ◽  
Anna Nitschke ◽  
Yannik Dreher ◽  
...  
Keyword(s):  
Lipid Vesicles ◽  
Cellular Systems ◽  
Top Down ◽  
Ph Gradients ◽  
Proton Gradients ◽  
Bottom Up ◽  
E Coli ◽  
Genetically Engineer ◽  
Synthetic Cells ◽  
Dna Triplex

Abstract Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness new types of living systems. Both approaches, however, face their own challenges towards biotechnological and biomedical applications. Here, we realize a strategic merger to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them as organelle mimics in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we can reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. By attaching a polymerized DNA origami plate to the DNA triplex motif, we obtain a cytoskeleton mimic that considerably deforms lipid vesicles in a pH-responsive manner. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells as potent microreactors.

scholarly journals Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wiggert J. Altenburg ◽  
N. Amy Yewdall ◽  
Daan F. M. Vervoort ◽  
Marleen H. M. E. van Stevendaal ◽  
Alexander F. Mason ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Nitrilotriacetic Acid ◽  
Binding Motif ◽  
Biologically Relevant ◽  
Enzyme Cascade ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Membrane Bound ◽  
High Concentrations ◽  
High Level

AbstractThe cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

scholarly journals Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics

2019 ◽  
Author(s):  
Patrick M. Caveney ◽  
Rosemary M. Dabbs ◽  
William T. McClintic ◽  
C. Patrick Collier ◽  
Michael L. Simpson
Keyword(s):  
Gene Expression ◽  
Membrane Transport ◽  
Molecular Species ◽  
Lipid Membranes ◽  
Molecular Transport ◽  
Lipid Vesicles ◽  
Cellular Systems ◽  
Control Of Gene Expression ◽  
External Resources ◽  
Synthetic Cell

SummaryEssential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7-fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.

scholarly journals Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kevin Jahnke ◽  
Noah Ritzmann ◽  
Julius Fichtler ◽  
Anna Nitschke ◽  
Yannik Dreher ◽  
...  
Keyword(s):  
Lipid Vesicles ◽  
Cellular Systems ◽  
Dna Origami ◽  
Top Down ◽  
Ph Gradients ◽  
Proton Gradients ◽  
Bottom Up ◽  
E Coli ◽  
Genetically Engineer ◽  
Synthetic Cells

AbstractBottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

scholarly journals Protein-free division of giant unilamellar vesicles controlled by enzymatic activity

2019 ◽  
Author(s):  
Yannik Dreher ◽  
Joachim P. Spatz ◽  
Kerstin Göpfrich
Keyword(s):  
Lipid Vesicles ◽  
Water Evaporation ◽  
Bottom Up ◽  
Life Success ◽  
Confocal Fluorescence ◽  
Synthetic Cells ◽  
Division Process ◽  
Physical Principles ◽  
Over Time ◽  
Time Point

AbstractCell division is one of the hallmarks of life. Success in the bottom-up assembly of synthetic cells will, no doubt, depend on strategies for the controlled autonomous division of protocellular compartments. Here, we describe the protein-free division of giant unilamellar lipid vesicles (GUVs) based on the combination of two physical principles – phase separation and osmosis. We visualize the division process with confocal fluorescence microscopy and derive a conceptual model based on the vesicle geometry. The model successfully predicts the shape transformations over time as well as the time point of the final pinching of the daughter vesicles. Remarkably, we show that two fundamentally distinct yet highly abundant processes – water evaporation and metabolic activity – can both regulate the autonomous division of GUVs. Our work may hint towards mechanisms that governed the division of protocells and adds to the strategic toolbox of bottom-up synthetic biology with its vision of bringing matter to life.

scholarly journals From primal scenes to synthetic cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Hub Zwart
Keyword(s):  
Synthetic Biology ◽  
Artificial Life ◽  
Living Cells ◽  
Philosophical Perspective ◽  
Fictional Narratives ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Life Projects

Synthetic cells spark intriguing questions about the nature of life. Projects such as BaSyC (Building a Synthetic Cell) aim to build an entity that mimics how living cells work. But what kind of entity would a synthetic cell really be? I assess this question from a philosophical perspective, and show how early fictional narratives of artificial life – such as the laboratory scene in Goethe’s Faust – can help us to understand the challenges faced by synthetic biology researchers.

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