scholarly journals Synthetic cell-based materials extract positional information from morphogen gradients

2021 ◽  
Author(s):  
Aurore Dupin ◽  
Lukas Aufinger ◽  
Igor Styazhkin ◽  
Florian Rothfischer ◽  
Benedikt Kaufmann ◽  
...  
Keyword(s):  
Expression System ◽  
Life Time ◽  
Positional Information ◽  
Lipid Bilayer Membranes ◽  
Gene Expression Noise ◽  
Morphogen Gradients ◽  
Emulsion Droplets ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Physical And Chemical

Dynamic biomaterials composed of synthetic cellular structures have the potential to adapt and functionally differentiate guided by physical and chemical cues from their environment. Inspired by developing biological systems, which efficiently extract positional information from chemical morphogen gradients in the presence of environmental uncertainties, we here investigate the analogous question: how well can a synthetic cell determine its position within a synthetic multicellular structure? In order to calculate positional information in such systems, we created and analyzed a large number of replicas of synthetic cellular assemblies, which were composed of emulsion droplets connected via lipid bilayer membranes. The droplets contained cell-free two-node feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. We found that in our system, simple anterior-posterior differentiation is possible, but positional information is limited by gene expression noise, and is also critically affected by the temporal evolution of the morphogen gradient and the life-time of the cell-free expression system contained in the synthetic cells. Using a 3D printing approach, we demonstrate morphogen-based differentiation also in larger tissue-like assemblies.

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 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 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 Gene expression noise in a complex artificial toxin expression system

PLoS ONE ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. e0227249
Author(s):  
Alexandra Goetz ◽  
Andreas Mader ◽  
Benedikt von Bronk ◽  
Anna S. Weiss ◽  
Madeleine Opitz
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Gene Expression Noise ◽  
Expression Noise

scholarly journals Dynamic positioning and precision of bistable gene expression boundaries through diffusion and morphogen decay

2020 ◽  
Author(s):  
Melinda Liu Perkins
Keyword(s):  
Gene Expression ◽  
Mathematical Theory ◽  
Initial Conditions ◽  
Propagation Speed ◽  
Positional Information ◽  
Future Research ◽  
Toggle Switch ◽  
Embryonic Patterning ◽  
Cell Coupling ◽  
Morphogen Gradients

1AbstractTraditional models for how morphogen gradients guide embryonic patterning fail to account for experimental observations of temporal refinement in gene expression domains. Dynamic positional information has recently emerged as a framework to address this shortcoming. Here, we explore two central aspects of dynamic positional information—the precision and placement of gene expression boundaries—in bistable genetic networks driven by morphogen gradients. First, we hypothesize that temporal morphogen decay may increase the precision of a boundary by compensating for variation in initial conditions that would otherwise lead neighboring cells with identical inputs to diverge to separate steady states. Second, we explore how diffusion of gene products may play a key role in placing gene expression boundaries. Using an existing model for Hb patterning in embryonic fruit flies, we show that diffusion permits boundaries to act as near-traveling wavefronts with local propagation speed determined by morphogen concentration. We then harness our understanding of near-traveling fronts to propose a method for achieving accurate steady-state boundary placement independent of initial conditions. Our work posits functional roles for temporally varying inputs and cell-to-cell coupling in the regulation and interpretation of dynamic positional information, illustrating that mathematical theory should serve to clarify not just our quantitative, but also our intuitive understanding of patterning processes.2Author SummaryIn many developmental systems, cells interpret spatial gradients of chemical morphogens to produce gene expression boundaries in exact positions. The simplest mathematical models for “positional information” rely on threshold detection, but such models are not robust to variations in the morphogen gradient or initial protein concentrations. Furthermore, these models fail to account for experimental results showing dynamic shifts in boundary placement and increased boundary precision over time. Here, we propose two theoretical mechanisms for enhancing boundary precision and placement using a bistable toggle switch. Distinct from existing research in “dynamic positional information”, this work posits a functional role for temporal decay in morphogen concentration and for diffusion of gene expression products, the latter of which is often omitted in quantitative models. We suggest that future research into dynamic positional information would benefit from perspectives that link local (cellular) and global (patterning) behaviors, as well as from mathematical theory that builds our intuitive understanding alongside more data-driven approaches.

scholarly journals MorphoGraphX 2.0: Providing context for biological image analysis with positional information

2021 ◽  
Author(s):  
Sören Strauss ◽  
Adam Runions ◽  
Brendan Lane ◽  
Dennis Eschweiler ◽  
Namrata Bajpai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Local Coordinate System ◽  
Growth Dynamics ◽  
Positional Information ◽  
Molecular Networks ◽  
Coordinate Systems ◽  
Morphogen Gradients ◽  
Generalized Framework ◽  
Plausible Mechanism ◽  
Microscopy Data

Positional information is a central concept in developmental biology. In developing organs, positional information can be idealized as a local coordinate system that arises from morphogen gradients controlled by organizers at key locations. This offers a plausible mechanism for the integration of the molecular networks operating in individual cells into the spatially-coordinated multicellular responses necessary for the organization of emergent forms. Understanding how positional cues guide morphogenesis requires the quantification of gene expression and growth dynamics in the context of their underlying coordinate systems. Here we present recent advances in the MorphoGraphX software (Barbier de Reuille et al. eLife 2015;4:e05864) that implement a generalized framework to annotate developing organs with local coordinate systems. These coordinate systems introduce an organ-centric spatial context to microscopy data, allowing gene expression and growth to be quantified and compared in the context of the positional information thought to control them.

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 CRISPR/Cas9-based Antibody Production in Plants

2020 ◽  
Vol 1 (2) ◽  
Author(s):  
Bareera Zahoor ◽  
Ummara Waheed ◽  
Shan Saeed ◽  
Fatima Gulzar ◽  
Hira Tasleem ◽  
...  
Keyword(s):  
Transient Expression ◽  
Antibody Production ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Production Plant ◽  
Post Translational Modification ◽  
Eukaryotic Expression System ◽  
Transient Expression System ◽  
Different Types ◽  
Physical And Chemical

Nowadays demand of antibody production is increased to cure different diseases including diabetes, hepatitis and cancer. For that different types of systems are used for the expression of antibody production. But these were not improved the antibody production. Plant cells have several benefits in comparison with other eukaryotic cells if it is considered as eukaryotic expression system. As compared to the human cell or other microorganisms, the plant cell is safe and decrease the contamination of antibody production. In addition, plants perform proper post-translational modification as a eukaryotic expression system. But recently, transient expression system is used due to the safe and improve the quality and quantity of antibody production. In transient expression system agroinfiltration method are mostly used. The main issue in antibody production is purification. Because in downstream process antibody is degraded due to the physical and chemical stresses. These issues can be solved with the help of CRISPR/Cas9. Plant antibody can be tagged with the help of CRISPR/Cas9. This review encompasses the applications of CRISPR technology for producing plant-based antibodies.

Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

2016 ◽  
Vol 22 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Filippo Caschera ◽  
Vincent Noireaux
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
Genetic Circuit ◽  
Translation System ◽  
Minimal Cell ◽  
E Coli ◽  
Synthetic Cell

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription–translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

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