scholarly journals Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

2016 ◽  
Vol 44 (3) ◽  
pp. 723-730 ◽  
Author(s):  
Yuval Elani
Keyword(s):  
Synthetic Biology ◽  
High Throughput ◽  
Lipid Membranes ◽  
Spatial Organization ◽  
Building Blocks ◽  
Artificial Cells ◽  
Bottom Up ◽  
Grand Challenges ◽  
New Frontier ◽  
Membrane Bound

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

scholarly journals Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Simon Bachler ◽  
Dominik Haidas ◽  
Marion Ort ◽  
Todd A. Duncombe ◽  
Petra S. Dittrich
Keyword(s):  
Synthetic Biology ◽  
Lipid Membranes ◽  
Bottom Up ◽  
Microfluidic Platform ◽  
Alpha Hemolysin ◽  
Droplet Interface Bilayer ◽  
Translocation Experiments ◽  
Fluorescent Molecules ◽  
Reaction Cascades ◽  
Minimal Cells

AbstractIn the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.

scholarly journals Microfluidics for Artificial Life: Techniques for Bottom-Up Synthetic Biology

Micromachines ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 299 ◽  
Author(s):  
Supramaniam ◽  
Ces ◽  
Salehi-Reyhani
Keyword(s):  
Synthetic Biology ◽  
Artificial Life ◽  
Living Cells ◽  
Cellular Biology ◽  
Artificial Cells ◽  
Bottom Up ◽  
Artificial Cell ◽  
Central Tenet ◽  
Cell Synthesis ◽  
New Generation

Synthetic biology is a rapidly growing multidisciplinary branch of science that exploits the advancement of molecular and cellular biology. Conventional modification of pre-existing cells is referred to as the top-down approach. Bottom-up synthetic biology is an emerging complementary branch that seeks to construct artificial cells from natural or synthetic components. One of the aims in bottom-up synthetic biology is to construct or mimic the complex pathways present in living cells. The recent, and rapidly growing, application of microfluidics in the field is driven by the central tenet of the bottom-up approach—the pursuit of controllably generating artificial cells with precisely defined parameters, in terms of molecular and geometrical composition. In this review we survey conventional methods of artificial cell synthesis and their limitations. We proceed to show how microfluidic approaches have been pivotal in overcoming these limitations and ushering in a new generation of complexity that may be imbued in artificial cells and the milieu of applications that result.

scholarly journals Enzyme-free synthesis of natural phospholipids in water

2019 ◽  
Author(s):  
Luping Liu ◽  
Yike Zou ◽  
Ahanjit Bhattacharya ◽  
Dongyang Zhang ◽  
Susan Q. Lang ◽  
...  
Keyword(s):  
Self Assembly ◽  
Soda Lakes ◽  
Building Blocks ◽  
Alkaline Solutions ◽  
Artificial Cells ◽  
Proton Gradients ◽  
Aqueous Synthesis ◽  
Acidic Solutions ◽  
Membrane Bound ◽  
Living Organisms

AbstractAll living organisms synthesize phospholipids as the primary constituent of their cell membranes. While phospholipids can spontaneously self-assemble in water to form membrane-bound vesicles, their aqueous synthesis requires pre-existing membrane-embedded enzymes. This limitation has led to models in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate that a combination of ion pairing and self-assembly of reactants allows high-yielding synthesis of cellular phospholipids in water. Acylation of 2-lysophospholipids using cationic thioesters occurs in mildly alkaline solutions resulting in the formation of cell-like membranes. A variety of membrane-forming natural phospholipids can be synthesized. Membrane formation takes place in water from natural alkaline sources, such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained.

scholarly journals Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

2018 ◽  
Vol 8 (5) ◽  
pp. 20180024 ◽  
Author(s):  
Tatiana Trantidou ◽  
Linda Dekker ◽  
Karen Polizzi ◽  
Oscar Ces ◽  
Yuval Elani
Keyword(s):  
Synthetic Biology ◽  
Genetically Engineered ◽  
Top Down ◽  
Giant Vesicles ◽  
Artificial Cells ◽  
Bottom Up ◽  
Regulatory Pathways ◽  
Genetically Engineered Microbes ◽  
Molecular Components ◽  
Cellular Components

The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes ( Escherichia coli ) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.

scholarly journals A Floating Mould Technique for the Programmed Assembly of Protocells into Protocellular Materials Capable of Non-Equilibrium Biochemical Sensing

2020 ◽  
Author(s):  
Agostino Galanti ◽  
Rafael Moreno Tortolero ◽  
Raihan Azad ◽  
Stephen Cross ◽  
Sean Davis ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Materials Science ◽  
Reaction Diffusion ◽  
Building Blocks ◽  
New Paradigm ◽  
Free Standing ◽  
Bottom Up ◽  
Periodic Arrays ◽  
Emergent Behaviours ◽  
Non Equilibrium

Despite important breakthroughs in bottom-up synthetic biology have recently been achieved, a major challenge still remains the construction of free-standing, macroscopic and robust materials from protocell building blocks that are stable in water and capable of emergent behaviours. Herein we report a new floating mould technique for the fabrication of millimetre- to centimetre-sized protocellular materials (PCMs) of any shape that overcomes most of the current challenges in prototissue engineering. Significantly, this technique also allowed us to generate 2D periodic arrays of PCMs that displayed an emergent non-equilibrium spatiotemporal sensing behaviour. These arrays were capable of collectively translating the information provided by the external environment and encoded in the form of propagating reaction-diffusion fronts into a readable dynamic signal output. Overall, our methodology opens up a route to the fabrication of macroscopicand robust tissue-like materials with emergent behaviours, providing a new paradigm of bottom-up synthetic biology and biomimetic materials science.

scholarly journals The interplay of spatial organization and biochemistry in building blocks of cellular signalling pathways

2020 ◽  
Vol 17 (166) ◽  
pp. 20200251 ◽  
Author(s):  
J. Krishnan ◽  
Lingjun Lu ◽  
Aiman Alam Nazki
Keyword(s):  
Information Processing ◽  
Synthetic Biology ◽  
Spatial Organization ◽  
Building Blocks ◽  
Covalent Modification ◽  
Model Organisms ◽  
Multiple Model ◽  
Biochemical Pathways ◽  
Two Component Systems

Biochemical pathways and networks are central to cellular information processing. While a broad range of studies have dissected multiple aspects of information processing in biochemical pathways, the effect of spatial organization remains much less understood. It is clear that space is central to intracellular organization, plays important roles in cellular information processing and has been exploited in evolution; additionally, it is being increasingly exploited in synthetic biology through the development of artificial compartments, in a variety of ways. In this paper, we dissect different aspects of the interplay between spatial organization and biochemical pathways, by focusing on basic building blocks of these pathways: covalent modification cycles and two-component systems, with enzymes which may be monofunctional or bifunctional. Our analysis of spatial organization is performed by examining a range of ‘spatial designs’: patterns of localization or non-localization of enzymes/substrates, theoretically and computationally. Using these well-characterized in silico systems, we analyse the following. (i) The effect of different types of spatial organization on the overall kinetics of modification, and the role of distinct modification mechanisms therein. (ii) How different information processing characteristics seen experimentally and studied from the viewpoint of kinetics are perturbed, or generated. (iii) How the activity of enzymes (bifunctional enzymes in particular) may be spatially manipulated, and the relationship between localization and activity. (iv) How transitions in spatial organization (encountered either through evolution or through the lifetime of cells, as seen in multiple model organisms) impacts the kinetic module (and pathway) behaviour, and how transitions in chemistry may be impacted by prior spatial organization. The basic insights which emerge are central to understanding the role of spatial organization in biochemical pathways in both bacteria and eukaryotes, and are of direct relevance to engineering spatial organization of pathways in bottom-up synthetic biology in cellular and cell-free systems.

scholarly journals A Floating Mould Technique for the Programmed Assembly of Protocells into Protocellular Materials Capable of Non-Equilibrium Biochemical Sensing

2020 ◽  
Author(s):  
Agostino Galanti ◽  
Rafael Moreno Tortolero ◽  
Raihan Azad ◽  
Stephen Cross ◽  
Sean Davis ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Materials Science ◽  
Reaction Diffusion ◽  
Building Blocks ◽  
New Paradigm ◽  
Free Standing ◽  
Bottom Up ◽  
Periodic Arrays ◽  
Emergent Behaviours ◽  
Non Equilibrium

Despite important breakthroughs in bottom-up synthetic biology have recently been achieved, a major challenge still remains the construction of free-standing, macroscopic and robust materials from protocell building blocks that are stable in water and capable of emergent behaviours. Herein we report a new floating mould technique for the fabrication of millimetre- to centimetre-sized protocellular materials (PCMs) of any shape that overcomes most of the current challenges in prototissue engineering. Significantly, this technique also allowed us to generate 2D periodic arrays of PCMs that displayed an emergent non-equilibrium spatiotemporal sensing behaviour. These arrays were capable of collectively translating the information provided by the external environment and encoded in the form of propagating reaction-diffusion fronts into a readable dynamic signal output. Overall, our methodology opens up a route to the fabrication of macroscopicand robust tissue-like materials with emergent behaviours, providing a new paradigm of bottom-up synthetic biology and biomimetic materials science.

scholarly journals Bioinspired Networks of Communicating Synthetic Protocells

2021 ◽  
Vol 8 ◽  
Author(s):  
Patrick J. Grimes ◽  
Agostino Galanti ◽  
Pierangelo Gobbo
Keyword(s):  
Tissue Engineering ◽  
Synthetic Biology ◽  
Regenerative Medicine ◽  
Living Cells ◽  
Chemical Signals ◽  
Biological Cells ◽  
Bottom Up ◽  
Grand Challenges ◽  
The Past

The bottom-up synthesis of cell-like entities or protocells from inanimate molecules and materials is one of the grand challenges of our time. In the past decade, researchers in the emerging field of bottom-up synthetic biology have developed different protocell models and engineered them to mimic one or more abilities of biological cells, such as information transcription and translation, adhesion, and enzyme-mediated metabolism. Whilst thus far efforts have focused on increasing the biochemical complexity of individual protocells, an emerging challenge in bottom-up synthetic biology is the development of networks of communicating synthetic protocells. The possibility of engineering multi-protocellular systems capable of sending and receiving chemical signals to trigger individual or collective programmed cell-like behaviours or for communicating with living cells and tissues would lead to major scientific breakthroughs with important applications in biotechnology, tissue engineering and regenerative medicine. This mini-review will discuss this new, emerging area of bottom-up synthetic biology and will introduce three types of bioinspired networks of communicating synthetic protocells that have recently emerged.

A Bottom-Up Approach to Solution-Processed, Atomically Precise Graphitic Cylinders on Surfaces

2018 ◽  
Author(s):  
Erik Leonhardt ◽  
Jeff M. Van Raden ◽  
David Miller ◽  
Lev N. Zakharov ◽  
Benjamin Aleman ◽  
...  
Keyword(s):  
Self Assembly ◽  
Carbon Nanostructures ◽  
Carbon Nanomaterials ◽  
Circle Packing ◽  
Pyrolytic Graphite ◽  
Building Blocks ◽  
Bottom Up ◽  
Casting Technique ◽  
New Class ◽  
Solution Casting Technique

Extended carbon nanostructures, such as carbon nanotubes (CNTs), exhibit remarkable properties but are difficult to synthesize uniformly. Herein, we present a new class of carbon nanomaterials constructed via the bottom-up self-assembly of cylindrical, atomically-precise small molecules. Guided by supramolecular design principles and circle packing theory, we have designed and synthesized a fluorinated nanohoop that, in the solid-state, self-assembles into nanotube-like arrays with channel diameters of precisely 1.63 nm. A mild solution-casting technique is then used to construct vertical “forests” of these arrays on a highly-ordered pyrolytic graphite (HOPG) surface through epitaxial growth. Furthermore, we show that a basic property of nanohoops, fluorescence, is readily transferred to the bulk phase, implying that the properties of these materials can be directly altered via precise functionalization of their nanohoop building blocks. The strategy presented is expected to have broader applications in the development of new graphitic nanomaterials with π-rich cavities reminiscent of CNTs.

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production

2019 ◽  
Author(s):  
Sean Lund ◽  
Taylor Courtney ◽  
Gavin Williams
Keyword(s):  
Synthetic Biology ◽  
Building Blocks ◽  
Isoprenoid Biosynthesis ◽  
Thermoplasma Acidophilum ◽  
Substrate Promiscuity ◽  
Enzymatic Pathways ◽  
First Time ◽  
Rate Limiting ◽  
Alcohol Dependent ◽  
Isopentenyl Phosphate

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in Nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene pathway (ADH) serve to leverage consecutive kinases to convert exogenous alcohols to pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway, isopentenyl phosphate kinase from Thermoplasma acidophilum, towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide non-natural pyrophosphates and provide a critical first step in characterizing the rate limiting steps in the artificial ADH pathway.<br>

Sign in / Sign up

Export Citation Format

Share Document