* joint first author # joint corresponding author

Katarzyna P Adamala#, Marileen Dogterom#, Yuval Elani#, Petra Schwille#, Masahiro Takinoue#, T-Y Dora Tang#
Present and future of synthetic cell development.
Nat Rev Mol Cell Biol, 25(3) 162-167 (2024)

Mengfei Gao, Dishi Wang, Michaela Wilsch-Bräuninger, Weihua Leng, Jonathan Schulte, Nina Morgner, Dietmar Appelhans, T-Y Dora Tang
Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.
Macromol Biosci, 24(3) Art. No. e2300464 (2024)
Open Access DOI
Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

Dishi Wang, Silvia Moreno#, Mengfei Gao, Jiaqi Guo, Bing Xu, Brigitte I Voit, Dietmar Appelhans#
Protocells Capable of Generating a Cytoskeleton-like Structure from intracellular Membrane-active Artificial Organelles.
Adv Funct Mater, 33(50) Art. No. 2306904 (2023)
Open Access DOI
The intricate nature of eukaryotic cells with differently viscous intracellular compartments provides (membrane-active) enzymes to trigger time- and concentration-dependent processes in the intra-/extracellular matrix. Herein, we capitalize on membrane-active artificial organelles (AOs) to develop fluidic and stable proteinaceous membrane-based protocells. AOs in protocells induce the self-assembly of oligopeptides into an artificial cytoskeleton that underline their influence on the structure and functionality of protocells. A series of microscopical tools is used to validate the intracellular assembly and distribution of cytoskeleton, the changing protocells morphology, and AOs inclusion within cytoskeletal growth. Thus, the dynamics, diffusion and viscosity of intracellular components in the presence of cytoskeleton are evaluated by fluorescence tools and enzymatic assay. Membrane-active alkaline phosphatase in polymersomes as AOs fulfills the requirements of biomimetic eukaryotic cells to trigger intracellular environment, mobility, viscosity, diffusion and enzymatic activity itself as well as high mechanical stability and high membrane fluidity of protocells. Thus membrane-active AOs in protocells thoroughly provide a variable reaction space in a changing intracellular environment and underline their regulatory role in the fabrication of complex protocell architectures and functions. This study demonstrates an important contribution to effective biomimicry of cell-like structures, shapes and functions.

T-Y Dora Tang#, Xin Huang#
Artificial Cells: From Basic Assembly to Directed Functionality.
Small Methods, 7(12) Art. No. e2301446 (2023)
Open Access DOI

David Thomas Gonzales, Surased Suraritdechachai, Christoph Zechner, T-Y Dora Tang
Bidirectional Communication between Droplet Interface Bilayers Driven by Cell-Free Quorum Sensing Gene Circuits.
ChemSystemsChem, 5 Art. No. e20230002 (2023)
Open Access DOI
Building synthetic multicellular systems using non-living molecular components is a grand challenge in the field of bottom-up synthetic biology. Towards this goal, a diverse range of chemistries have been developed to provide mechanisms of intercellular communication and methods to assemble multicellular compartments. However, building bottom-up synthetic multicellular systems is still challenging because it requires the integration of intercellular reaction networks with compatible cellular compartment properties. In this study, we encapsulated cell-free expression systems (CFES) expressing two quorum sensing genetic circuits into droplet interface bilayer (DIB) synthetic cells to demonstrate bidirectional communication. We further develop a method of generating custom DIB multicellular structures by acoustic liquid handling to automatically dispense the CFES droplets and show the potential for multiplexing compartmentalized gene circuits for generating heterogeneous populations of cells. Our work provides a step towards building more complex multicellular systems with intercellular communication from the bottom-up to study and experimentally model biological multiscalar processes.

Kristian Le Vay✳︎#, Elia Salibi✳︎, Basusree Ghosh, T Y Dora Tang#, Hannes Mutschler#
Ribozyme activity modulates the physical properties of RNA-peptide coacervates.
Elife, 12 Art. No. e83543 (2023)
Open Access DOI
Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

David Thomas Gonzales, Surased Suraritdechachai, T Y Dora Tang
Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells.
Adv Biochem Eng Biotechnol, 186 77-101 (2023)
One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

Nino Lauber, Ondrej Tichacek, Rudrarup Bose, Christoph Flamm, Luca Leuzzi, T-Y Dora Tang, Kepa Ruiz-Mirazo, Daniele De Martino
Statistical mechanics of biomolecular condensates via cavity methods.
iScience, 26(4) Art. No. 106300 (2023)
Open Access DOI
Physical mechanisms of phase separation in living systems play key physiological roles and have recently been the focus of intensive studies. The strongly heterogeneous nature of such phenomena poses difficult modeling challenges that require going beyond mean-field approaches based on postulating a free energy landscape. The pathway we take here is to calculate the partition function starting from microscopic interactions by means of cavity methods, based on a tree approximation for the interaction graph. We illustrate them on the binary case and then apply them successfully to ternary systems, in which simpler one-factor approximations are proved inadequate. We demonstrate the agreement with lattice simulations and contrast our theory with coacervation experiments of associative de-mixing of nucleotides and poly-lysine. Different types of evidence are provided to support cavity methods as ideal tools for modeling biomolecular condensation, giving an optimal balance between the consideration of spatial aspects and fast computational results.

Christine Mayr, Tanja Mittag, T-Y Dora Tang, Wenyu Wen, Hong Zhang, Huaiying Zhang
Frontiers in biomolecular condensate research.
Nat Cell Biol, 25(4) 512-514 (2023)

Xuejing Wang, Shuqi Wu, T-Y Dora Tang, Liangfei Tian
Engineering strategies for sustainable synthetic cells.
Trends in Chemistry , 4(12) 1106-1120 (2022)
An ongoing grand challenge in bottom-up synthetic biology is designing and constructing synthetic cells with life-like properties. Despite the significant advances, even the most highly integrated synthetic cell does not come close to living entities. One of the main differences is that biological cells are dynamic and show self-regulating behavior, while synthetic cellular mimics will eventually reach a static state and exhibit unidirectional responses to perturbation. To this end, the next milestone for bottom-up synthetic biology will be the development of sustainable synthetic cellular systems that can show functional dynamic behavior. Here, we review the engineering strategies to design and construct such functional synthetic cellular systems and the challenges and potential future opportunities in this field are discussed.

Adrian Zambrano✳︎, Giorgio Fracasso✳︎, Mengfei Gao✳︎, Martina Ugrinic, Dishi Wang, Dietmar Appelhans, Andrew deMello, T-Y Dora Tang
Programmable synthetic cell networks regulated by tuneable reaction rates.
Nat Commun, 13(1) Art. No. 3885 (2022)
Open Access DOI
Coupled compartmentalised information processing and communication via molecular diffusion underpin network based population dynamics as observed in biological systems. Understanding how both compartmentalisation and communication can regulate information processes is key to rational design and control of compartmentalised reaction networks. Here, we integrate PEN DNA reactions into semi-permeable proteinosomes and characterise the effect of compartmentalisation on autocatalytic PEN DNA reactions. We observe unique behaviours in the compartmentalised systems which are not accessible under bulk conditions; for example, rates of reaction increase by an order of magnitude and reaction kinetics are more readily tuneable by enzyme concentrations in proteinosomes compared to buffer solution. We exploit these properties to regulate the reaction kinetics in two node compartmentalised reaction networks comprised of linear and autocatalytic reactions which we establish by bottom-up synthetic biology approaches.

Damian Wollny#, Benjamin Vernot, Jie Wang, Maria Hondele, Aram Safrastyan, Franziska Aron, Julia Micheel, Zhisong He, Anthony Hyman, Karsten Weis, J Gray Camp, T-Y Dora Tang#, Barbara Treutlein#
Characterization of RNA content in individual phase-separated coacervate microdroplets.
Nat Commun, 13(1) Art. No. 2626 (2022)
Open Access DOI
Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Juan M Iglesias-Artola, Björn Drobot, Mrityunjoy Kar, Anatol Fritsch, Hannes Mutschler, T-Y Dora Tang, Moritz Kreysing
Charge-density reduction promotes ribozyme activity in RNA-peptide coacervates via RNA fluidization and magnesium partitioning.
Nat Chem, 14(4) 407-416 (2022)
Open Access PDF DOI
It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA-peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

Dora Tang
Cell scientist to watch - Dora Tang.
J Cell Sci, 135(5) Art. No. jcs259851 (2022)
Dora Tang studied chemistry and then completed her PhD in membrane physics at Imperial College London, UK, under the supervision of John Seddon and Richard Templer. She then received Knowledge Transfer Secondment funding from Imperial College and worked for a year at Diamond Light Source, Oxfordshire, the UK's national synchrotron science facility. In 2011, Dora joined the lab of Stephen Mann at the University of Bristol, where she worked in the areas of the origin of life and bottom-up synthetic biology. Her research there included developing a hybrid protocell model based on the self-assembly of fatty acid membrane on coacervate microdroplets, as well as showing that coacervates support cell-free gene expression and building communication networks between two different populations of synthetic cells. In 2016, she started her own group at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, as part of the MaxSynBio consortium. Her research aims to understand the chemical and physical processes that drive molecular organisation in lipids, polymers and proteins in order to rationally control their self-assembly and understand how molecular ensembles make life.

David Thomas Gonzales, Naresh Yandrapalli, Tom Robinson, Christoph Zechner#, T-Y Dora Tang#
Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.
ACS Synth Biol, 11(1) 205-215 (2022)
Open Access DOI
The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

Alan Ianeselli, Damla Tetiker, Julian Stein, Alexandra Kühnlein, Christof Mast, Dieter Braun#, T-Y Dora Tang#
Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of coacervate protocells.
Nat Chem, 14(1) 32-39 (2022)
Open Access DOI
Key requirements for the first cells on Earth include the ability to compartmentalize and evolve. Compartmentalization spatially localizes biomolecules from a dilute pool and an evolving cell, which, as it grows and divides, permits mixing and propagation of information to daughter cells. Complex coacervate microdroplets are excellent candidates as primordial cells with the ability to partition and concentrate molecules into their core and support primitive and complex biochemical reactions. However, the evolution of coacervate protocells by fusion, growth and fission has not yet been demonstrated. In this work, a primordial environment initiated the evolution of coacervate-based protocells. Gas bubbles inside heated rock pores perturb the coacervate protocell distribution and drive the growth, fusion, division and selection of coacervate microdroplets. Our findings provide a compelling scenario for the evolution of membrane-free coacervate microdroplets on the early Earth, induced by common gas bubbles within heated rock pores.

Kristian Le Vay✳︎#, Emilie Yeonwha Song✳︎, Basusree Ghosh, T-Y Dora Tang#, Hannes Mutschler#
Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.
Angew Chem Int Ed Engl, 60(50) 26096-26104 (2021)
Open Access DOI
The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.

Lars Hubatsch, Louise Jawerth, Celina Love, Jonathan Bauermann, Ty Dora Tang, Stefano Bo, Anthony Hyman, Christoph A. Weber
Quantitative theory for the diffusive dynamics of liquid condensates.
Elife, 10 Art. No. e68620 (2021)
Open Access DOI
Key processes of biological condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date, a physics-based, quantitative framework for the dynamics of labeled condensate components is lacking. Here we derive the corresponding dynamic equations, building on the physics of phase separation, and quantitatively validate the related framework via experiments. We show that by using our framework we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Interestingly, our theory can also be used to determine a relationship between the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This enables us to investigate the effect of salt addition on partitioning and bypasses recently described quenching artifacts in the dense phase. Our approach opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities, and quantifying rates of biochemical reactions in liquid condensates.

Basusree Ghosh✳︎, Rudrarup Bose✳︎, T-Y Dora Tang
Can coacervation unify disparate hypotheses in the origin of cellular life?
Curr Opin Colloid Interface Sci, 52 Art. No. 101415 (2021)
Open Access DOI

Kristian Le Vay, Ben M Carter, Daniel W Watkins, T-Y Dora Tang, Valeska P Ting, Helmut Cölfen, Robert P Rambo, Andrew J Smith, J L Ross Anderson, Adam W Perriman
Controlling Protein Nanocage Assembly with Hydrostatic Pressure.
J Am Chem Soc, 142(49) 20640-20650 (2020)
Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalization of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron radiation small-angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr 24-mer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the interdimer interface that is key to cage assembly. This indicates a major cage-stabilizing role for heme within this family of ferritins.

Nicolette G Moreau, Nicolas Martin, Pierangelo Gobbo, T-Y Dora Tang, Stephen Mann
Spontaneous membrane-less multi-compartmentalization via aqueous two-phase separation in complex coacervate micro-droplets.
Chem Commun (Camb), 56(84) 12717-12720 (2020)
Open Access DOI
Polyelectrolyte/nucleotide multiphase complex coacervate droplets are produced by internalized aqueous two-phase separation and used for the spatially dependent chemical transfer of sugar molecules, providing a step towards the development of membrane-free "organelles" within coacervate-based protocells.

David Thomas Gonzales, Christoph Zechner, T-Y Dora Tang
Building synthetic multicellular systems usingbottom–up approaches.
Curr Opin Syst Biol, 24 56-63 (2020)
Open Access DOI
A grand challenge in bottom–up synthetic biology is the designand construction of synthetic multicellular systems usingnonliving molecular components. Abstracting key features ofcompartmentalisation, reaction and diffusion, and communica-tion provides the blueprint for assembling synthetic multiscalesystems with emergent properties. The diverse range of chem-istries for building encapsulated reactions in micron-sized com-partments offers combinatorial flexibility and modularity inbuilding synthetic multicellular systems with molecular-levelcontrol. Here, we focus onrecent advancesin the emergingareaof bottom–up approaches to create biologically inspired multi-cellular systems. Specifically, we consider how intercellularcommunication and feedback loops can be integrated intopopulations of synthetic cells and summarise recent de-velopments for the 2D/3D spatial localisation of microcompart-ments. Although building bottom–upmulticellular systems isstillin its infancy, progress in this field offers tractable models to un-derstand the minimal requirements for generating multiscalesystems from the molecular level for fundamental research andinnovative technological applications.

Thomas E. Gorochowski, Sabine Hauert, Jan-Ulrich Kreft, Lucia Marucci, Namid R. Stillman, T-Y Dora Tang, Lucia Bandiera, Vittorio Bartoli, Daniel O.R. Dixon, Alex J.H. Fedorec, Harold Fellermann, Alexander G. Fletcher, Tim Foster, Luca Giuggioli, Antoni Matyjaszkiewicz, Scott McCormick, Sandra Montes Olivas, Jonathan Naylor, Ana Rubio Denniss, Daniel Ward
Toward Engineering Biosystems With Emergent Collective Functions.
Front Bioeng Biotechnol, 8 Art. No. 705 (2020)
Open Access DOI
Many complex behaviors in biological systems emerge from large populations of interacting molecules or cells, generating functions that go beyond the capabilities of the individual parts. Such collective phenomena are of great interest to bioengineers due to their robustness and scalability. However, engineering emergent collective functions is difficult because they arise as a consequence of complex multi-level feedback, which often spans many length-scales. Here, we present a perspective on how some of these challenges could be overcome by using multi-agent modeling as a design framework within synthetic biology. Using case studies covering the construction of synthetic ecologies to biological computation and synthetic cellularity, we show how multi-agent modeling can capture the core features of complex multi-scale systems and provide novel insights into the underlying mechanisms which guide emergent functionalities across scales. The ability to unravel design rules underpinning these behaviors offers a means to take synthetic biology beyond single molecules or cells and toward the creation of systems with functions that can only emerge from collectives at multiple scales.

Thomas Beneyton✳︎, Celina Love✳︎, Mathias Girault, T-Y Dora Tang#, Jean-Christophe Baret#
High-Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics.
ChemSystemsChem, 2(6) Art. No. e2000022 (2020)
Open Access DOI
To understand how membrane‐free subcompartmentalization can modulate biochemical reactions by coupled spatial enzyme localization with substrate and product partitioning, we use microfluidic strategies to synthesize, stabilize and characterize micron‐sized functional coacervates in water−oil emulsions. Our methodologies have allowed for the first time to quantitatively characterize partition coefficients of a broad range of different molecules with different coacervate chemistries and to measure reaction rates of individual subcompartments and their surrounding aqueous environment at the single coacervate level. Our results show that sub‐compartmentalisation increases the overall rates of reactions. This bottom‐up synthetic strategy for the production of synthetic organelles offers a physical model for membrane‐free compartmentalization in biology and provides insights into the role of sub‐compartmentalisation in regulating out‐of‐equilibrium behaviours in biological systems.

Celina Love, Jan Steinkühler, David Thomas Gonzales, Naresh Yandrapalli, Tom Robinson, Rumiana Dimova, T-Y Dora Tang
Reversible pH-Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions.
Angew Chem Int Ed Engl, 59(15) 5950-5957 (2020)
Open Access DOI
In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pKa , to drive liquid-liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pKa . We further show that pH-triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi-compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom-up synthetic biology approach.

David Thomas Gonzales, T-Y Dora Tang, Christoph Zechner
Moment-based analysis of biochemical networks in a heterogeneous population of communicating cells.
In: 2019 IEEE 58th Conference on Decision and Control (CDC) (2019)(Eds.) Carlos A. Canudas de Wit, Piscataway, N.J., IEEE (2019), 939-944
Cells can utilize chemical communication to exchange information and coordinate their behavior in the presence of noise. Communication can reduce noise to shape a collective response, or amplify noise to generate distinct phenotypic subpopulations. Here we discuss a moment-based approach to study how cell-cell communication affects noise in biochemical networks that arises from both intrinsic and extrinsic sources. We derive a system of approximate differential equations that captures lower-order moments of a population of cells, which communicate by secreting and sensing a diffusing molecule. Since the number of obtained equations grows combinatorially with number of considered cells, we employ a previously proposed model reduction technique, which exploits symmetries in the underlying moment dynamics. Importantly, the number of equations obtained in this way is independent of the number of considered cells such that the method scales to arbitrary population sizes. Based on this approach, we study how cell-cell communication affects population variability in several biochemical networks. Moreover, we analyze the accuracy and computational efficiency of the moment-based approximation by comparing it with moments obtained from stochastic simulations.

Hannes Mutschler, Tom Robinson, T-Y Dora Tang, Seraphine Wegner
Special Issue on Bottom-Up Synthetic Biology.
Chembiochem, 20(20) 2533-2534 (2019)
Open Access DOI
Bottom-up synthetic biology uses both biological and artificial chemical building blocks to create biomimetic systems, including artificial cells. Existing and new technologies such as microfluidics are being developed and applied to the assembly processes. In this special issue, experts present and review the latest progress in this rapidly expanding and diverse field.

Martina Ugrinic, Andrew deMello#, T-Y Dora Tang#
Microfluidic Tools for Bottom-Up Synthetic Cellularity.
Chem, 5(7) 1727-1742 (2019)

Giorgio Fracasso, Yvonne Körner, David Thomas Gonzales, T-Y Dora Tang
In vitro gene expression and detergent-free reconstitution of active proteorhodopsin in lipid vesicles.
Exp Biol Med (Maywood), 244(4) 314-322 (2019)
Our results offer the potential for straightforward, additive-free, and molecularly simple routes to building complex bioreactors based on in vitro transcription-translation systems and lipid vesicles.

Ivan Ivanov#, Rafael B. Lira, T-Y Dora Tang, Titus Franzmann, Adam Klosin, Lucas Caire da Silva, Anthony Hyman, Katharina Landfester, Reinhard Lipowsky, Kai Sundmacher, Rumiana Dimova#
Directed Growth of Biomimetic Microcompartments.
Adv Biosys, 3(6) Art. No. 1800314 (2019)

Petra Schwille, Joachim P. Spatz, Katharina Landfester, Eberhard Bodenschatz, Stephan Herminghaus, Victor Sourjik, Tobias Erb, Philippe Bastiaens, Reinhard Lipowsky, Anthony Hyman, Peter Dabrock, Jean-Christophe Baret, Tanja Vidakovic-Koch, Peter Bieling, Rumiana Dimova, Hannes Mutschler, Tom Robinson, T-Y Dora Tang, Seraphine Wegner, Kai Sundmacher
MaxSynBio: Avenues Towards Creating Cells from the Bottom Up.
Angew Chem Int Ed Engl, 57(41) 13382-13392 (2018)
A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

Björn Drobot, Juan M Iglesias-Artola, Kristian Le Vay, Viktoria Mayr, Mrityunjoy Kar, Moritz Kreysing#, Hannes Mutschler#, T-Y Dora Tang#
Compartmentalised RNA catalysis in membrane-free coacervate protocells.
Nat Commun, 9(1) Art. No. 3643 (2018)
Open Access PDF DOI
Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

Toni Haubitz, Satoru Tsushima, Robin Steudtner, Björn Drobot, Gerhard Geipel, Thorsten Stumpf, Michael U. Kumke
Ultrafast Transient Absorption Spectroscopy of UO22+ and [UO2Cl].
J Phys Chem A, 122(35) 6970-6977 (2018)
For the only water coordinated "free" uranyl(VI) aquo ion in perchlorate solution we identified and assigned several different excited states and showed that the 3Δ state is the luminescent triplet state from transient absorption spectroscopy. With additional data from other spectroscopic methods (TRLFS, UV/vis) we generated a detailed Jabłoński diagram and determined rate constants for several state transitions, like the inner conversion rate constant from the 3Φ state to the 3Δ state transition to be 0.35 ps-1. In contrast to luminescence measurements, it was possible to observe the highly quenched uranyl(VI) ion in highly concentrated chloride solution by TAS and we were able to propose a dynamic quenching mechanism, where chloride complexation is followed by the charge transfer from the excited state uranyl(VI) to chloride. This proposed quenching route is supported by TD-DFT calculations.

T-Y Dora Tang, Dario Cecchi, Giorgio Fracasso, Davide Accardi, Angelique Coutable-Pennarun, Sheref S Mansy, Adam W Perriman, J L Ross Anderson, Stephen Mann
Gene-Mediated Chemical Communication in Synthetic Protocell Communities.
ACS Synth Biol, 7(2) 339-346 (2018)
A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substrate signaling, and enzyme cascade-mediated processing.

Martina Ugrinic, Adrian Zambrano, Simon Berger, Stephen Mann, T-Y Dora Tang, Andrew deMello
Microfluidic formation of proteinosomes.
Chem Commun (Camb), 54(3) 287-290 (2018)
Open Access DOI
Herein we describe a novel microfluidic method for the generation of proteinosome micro-droplets, based on bovine serum albumin and glucose oxidase conjugated to PNIPAAm chains. The size of such water-in-oil droplets is regulated via control of the input reagent flow rate, with generated proteinosome populations exhibiting narrower size distributions than those observed when using standard bulk methodologies. Importantly, proteinosomes transferred from an oil to an aqueous-environment remain intact, become fully hydrated and exhibit an increase in average size. Moreover, functional proteinosomes prepared via microfluidics exhibit lower Km values and higher enzymatic activities than proteinosomes produced by bulk methodologies.

Jeffrey R Vieregg, T-Y Dora Tang
Polynucleotides in cellular mimics: Coacervates and lipid vesicles
Curr Opin Colloid Interface Sci, 26 50-57 (2016)

Jennifer M Bulpett, Tim Snow, Benoit Quignon, Charlotte M Beddoes, T-Y D Tang, Stephen Mann, Olga Shebanova, Claire L Pizzey, Nicholas J Terrill, Sean A Davis, Wuge H Briscoe
Hydrophobic nanoparticles promote lamellar to inverted hexagonal transition in phospholipid mesophases.
Soft Matter, 11(45) 8789-8800 (2015)
This study focuses on how the mesophase transition behaviour of the phospholipid dioleoyl phosphatidylethanolamine (DOPE) is altered by the presence of 10 nm hydrophobic and 14 nm hydrophilic silica nanoparticles (NPs) at different concentrations. The lamellar to inverted hexagonal phase transition (Lα-HII) of phospholipids is energetically analogous to the membrane fusion process, therefore understanding the Lα-HII transition with nanoparticulate additives is relevant to how membrane fusion may be affected by these additives, in this case the silica NPs. The overriding observation is that the HII/Lα boundaries in the DOPE p-T phase diagram were shifted by the presence of NPs: the hydrophobic NPs enlarged the HII phase region and thus encouraged the inverted hexagonal (HII) phase to occur at lower temperatures, whilst hydrophilic NPs appeared to stabilise the Lα phase region. This effect was also NP-concentration dependent, with a more pronounced effect for higher concentration of the hydrophobic NPs, but the trend was less clear cut for the hydrophilic NPs. There was no evidence that the NPs were intercalated into the mesophases, and as such it was likely that they might have undergone microphase separation and resided at the mesophase domain boundaries. Whilst the loci and exact roles of the NPs invite further investigation, we tentatively discuss these results in terms of both the surface chemistry of the NPs and the effect of their curvature on the elastic bending energy considerations during the mesophase transition.

T-Y Dora Tang, Dirk van Swaay, Andrew deMello, J L Ross Anderson, Stephen Mann
In vitro gene expression within membrane-free coacervate protocells.
Chem Commun (Camb), 51(57) 11429-11432 (2015)
Cell-free gene expression of a fluorescent protein (mCherry) is demonstrated within the molecularly crowded matrix of a polysaccharide/polypeptide coacervate.

Dirk van Swaay, T-Y Dora Tang, Stephen Mann, Andrew de Mello
Microfluidic Formation of Membrane-Free Aqueous Coacervate Droplets in Water.
Angew Chem Int Ed Engl, 54(29) 8398-8401 (2015)
We report on the formation of coacervate droplets from poly(diallyldimethylammonium chloride) with either adenosine triphosphate or carboxymethyl-dextran using a microfluidic flow-focusing system. The formed droplets exhibit improved stability and narrower size distributions for both coacervate compositions when compared to the conventional vortex dispersion techniques. We also demonstrate the use of two parallel flow-focusing channels for the simultaneous formation and co-location of two distinct populations of coacervate droplets containing different DNA oligonucleotides, and that the populations can coexist in close proximity up to 48 h without detectable exchange of genetic information. Our results show that the observed improvements in droplet stability and size distribution may be scaled with ease. In addition, the ability to encapsulate different materials into coacervate droplets using a microfluidic channel structure allows for their use as cell-mimicking compartments.

T-Y Dora Tang, Nicholas J Brooks, Oscar Ces, John M Seddon, Richard H Templer
Structural studies of the lamellar to bicontinuous gyroid cubic (Q(G)(II)) phase transitions under limited hydration conditions.
Soft Matter, 11(10) 1991-1997 (2015)
Non-equilibrium pathways of lyotropic phase transitions such as the lamellar to inverse bicontinuous cubic phase transition are important dynamical processes resembling cellular fusion and fission processes which can be exploited in biotechnological processes such as drug delivery. However, utilising and optimising these structural transformations for applications require a detailed understanding of the energetic pathways which drive the phase transition. We have used the high pressure X-ray diffraction technique to probe the lamellar to Q(G)(II) phase transition in limited hydration monolinolein on the millisecond time scale. Our results show that the phase transition goes via a structural intermediate and once the Q(G)(II) phase initially forms the elastic energy in the bilayer drives this structure to its equilibrium lattice parameter.

Mei Li, Xin Huang, T-Y Dora Tang, Stephen Mann
Synthetic cellularity based on non-lipid micro-compartments and protocell models.
Curr Opin Chem Biol, 22 1-11 (2014)
This review discusses recent advances in the design and construction of protocell models based on the self-assembly or microphase separation of non-lipid building blocks. We focus on strategies involving partially hydrophobic inorganic nanoparticles (colloidosomes), protein-polymer globular nano-conjugates (proteinosomes), amphiphilic block copolymers (polymersomes), and stoichiometric mixtures of oppositely charged biomolecules and polyelectrolytes (coacervates). Developments in the engineering of membrane functionality to produce synthetic protocells with gated responses and control over multi-step reactions are described. New routes to protocells comprising molecularly crowded, cytoskeletal-like hydrogel interiors, as well as to the construction of hybrid protocell models are also highlighted. Together, these strategies enable a wide range of biomolecular and synthetic components to be encapsulated, regulated and processed within the micro-compartmentalized volume, and suggest that the development of non-lipid micro-ensembles offers an approach that is complementary to protocell models based on phospholipid or fatty acid vesicles.

T-Y Dora Tang, C Rohaida Che Hak, Alexander J Thompson, Marina K Kuimova, D S Williams, Adam W Perriman, Stephen Mann
Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model.
Nat Chem, 6(6) 527-533 (2014)
Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes.

T-Y Dora Tang, Annela M Seddon, Christoph Jeworrek, Roland Winter, Oscar Ces, John M Seddon, Richard H Templer
The effects of pressure and temperature on the energetics and pivotal surface in a monoacylglycerol/water gyroid inverse bicontinuous cubic phase.
Soft Matter, 10(17) 3009-3015 (2014)
We have studied the effect of pressure and temperature on the location of the pivotal surface in a lipid inverse bicontinuous gyroid cubic phase (Q(G)(II)), described by the area at the pivotal surface (An), the volume between the pivotal surface and the bilayer midplane (Vn), and the molecular volume of the lipid (V). Small angle X-ray scattering (SAXS) was used to measure the swelling behaviour of the lipid, monolinolein, as a function of pressure and temperature, and the data were fitted to two different geometric models: the parallel interface model (PIM), and the constant mean curvature model (CMCM). The results show that an increase in temperature leads to a shift in the location of the pivotal surface towards the bilayer midplane, whilst an increase in pressure causes the pivotal surface to move towards the interfacial region. In addition, we describe the relevance of An, Vn and V for modeling the energetics of curved mesophases with specific reference to the mean curvature at the pivotal surface and discuss the significance of this parameter for modelling the energetics of curved mesophases.

T-Y Dora Tang, Massimo Antognozzi, James A. Vicary, Adam W Perriman, Stephen Mann
Small-molecule uptake in membrane-free peptide/nucleotide protocells
Soft Matter, 9 7647-7656 (2013)

T-Y Dora Tang, Nicholas J Brooks, Christoph Jeworrek, Oscar Ces, Nicholas J Terrill, Roland Winter, Richard H Templer, John M Seddon
Hydrostatic pressure effects on the lamellar to gyroid cubic phase transition of monolinolein at limited hydration.
Langmuir, 28(36) 13018-13024 (2012)
Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.

Chandrashekhar V. Kulkarni, T-Y Dora Tang, Annela M Seddon, John M Seddon, Oscar Ces, Richard H Templer
Engineering bicontinuous cubic structures at the nanoscale—the role of chain splay
Soft Matter, 6 3191-3194 (2010)