A microfluidic platform for sequential assembly and separation of synthetic cell models

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and by-products, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a stream of vesicles suspension is partitioned into three fractions - purified GUVs, residual components, and a washing solution. Using our purification approach, we showed that giant vesicles can be separated from various residues, that range in size and chemical composition, with a very high efficiency (e=0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

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From primal scenes to synthetic cells

eLife ◽

10.7554/elife.46518 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

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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Synthetic Cells Engaged in Molecular Communication: An Opportunity for Modelling Shannon- and Semantic-Information in the Chemical Domain

Frontiers in Communications and Networks ◽

10.3389/frcmn.2021.724597 ◽

2021 ◽

Vol 2 ◽

Author(s):

Maurizio Magarini ◽

Pasquale Stano

Keyword(s):

Synthetic Biology ◽

Semantic Information ◽

Emerging Technology ◽

Shannon Information ◽

Molecular Communication ◽

Bottom Up ◽

Nano Scale ◽

Synthetic Cell ◽

Synthetic Cells ◽

Cell Fabrication

In this Perspective article we intend to focus on the opportunity of modelling Shannon information and/or “semantic” information in the field originated by the convergence of bottom-up synthetic biology (in particular, the construction of “synthetic cells”) and the engineering approaches to molecular communication. In particular we will argue that the emerging technology of synthetic cell fabrication will allow novel opportunities to study nano-scale communication and manipulation of information in unprecedented manner. More specifically, we will discuss the possibility of enquiring on the transfer and manipulation of information in the chemical domain, and interpreting such a dynamics according to Shannon or to MacKay-Bateson (“semantic” information).

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A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models

ACS Synthetic Biology ◽

10.1021/acssynbio.1c00371 ◽

2021 ◽

Author(s):

Ran Tivony ◽

Marcus Fletcher ◽

Kareem Al Nahas ◽

Ulrich F. Keyser

Keyword(s):

Cell Models ◽

Microfluidic Platform ◽

Synthetic Cell ◽

Sequential Assembly

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CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Vecchione ◽

Georg Fritz

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Integrated Circuits ◽

High Efficiency ◽

Genetic Circuit ◽

Dna Assembly ◽

Chromosomal Integration ◽

Golden Gate ◽

Genetic Circuits ◽

E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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

Communications Biology ◽

10.1038/s42003-020-01489-w ◽

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.

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A Circular Gradient-Width Crossflow Microfluidic Platform for High-Efficiency Blood Plasma Separation

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2021.131180 ◽

2021 ◽

pp. 131180

Author(s):

Han Zhang ◽

Kanjirakat Anoop ◽

Can Huang ◽

Reza Sadr ◽

Rohit Gupte ◽

...

Keyword(s):

Blood Plasma ◽

High Efficiency ◽

Microfluidic Platform ◽

Plasma Separation ◽

Blood Plasma Separation

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Investigating Prebiotic Protocells for a Comprehensive Understanding of the Origins of Life: A Prebiotic Systems Chemistry Perspective

Life ◽

10.3390/life9020049 ◽

2019 ◽

Vol 9 (2) ◽

pp. 49 ◽

Cited By ~ 9

Author(s):

Augustin Lopez ◽

Michele Fiore

Keyword(s):

Thin Films ◽

Synthetic Biology ◽

Chemical Compounds ◽

Origins Of Life ◽

Supramolecular Systems ◽

Comprehensive Understanding ◽

Giant Vesicles ◽

Relevant Concept ◽

Systems Chemistry ◽

Emergence Of Life

Protocells are supramolecular systems commonly used for numerous applications, such as the formation of self-evolvable systems, in systems chemistry and synthetic biology. Certain types of protocells imitate plausible prebiotic compartments, such as giant vesicles, that are formed with the hydration of thin films of amphiphiles. These constructs can be studied to address the emergence of life from a non-living chemical network. They are useful tools since they offer the possibility to understand the mechanisms underlying any living cellular system: Its formation, its metabolism, its replication and its evolution. Protocells allow the investigation of the synergies occurring in a web of chemical compounds. This cooperation can explain the transition between chemical (inanimate) and biological systems (living) due to the discoveries of emerging properties. The aim of this review is to provide an overview of relevant concept in prebiotic protocell research.

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Ultra-high capacity microfluidic trapping of giant vesicles for high-throughput membrane studies

Lab on a Chip ◽

10.1039/c8lc01275j ◽

2019 ◽

Vol 19 (4) ◽

pp. 626-633 ◽

Cited By ~ 14

Author(s):

Naresh Yandrapalli ◽

Tom Robinson

Keyword(s):

High Throughput ◽

High Capacity ◽

Giant Vesicles ◽

Microfluidic Platform

A high-capacity microfluidic platform designed to capture tens of thousands of giant vesicles for high-throughput membrane analysis.

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

Nature Communications ◽

10.1038/s41467-020-20124-0 ◽

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.

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