Biology subjects

Danelon, C.

Publications and source records attributed to Danelon, C..

16 recordsLinked to original sources

bioRxiv2026

Optimization of PURE system composition using automation and active learning

Protein synthesis using recombinant elements (PURE) system has been widely applied in various biological research fields and synthetic cell construction. Optimization efforts to enhance the PURE system performance by adjusting its individual components have remained limited to the expression of single genes with a small number of molecular compositions tested, making it difficult to link component composition to system-level performance across different DNA contexts. Here, we combine automated acoustic liquid handling with an active learning framework to explore broadly the compositional landscape of PURE system. By grouping the 69 individual components (including proteins and tRNAs) into 21 functional sets and iteratively guiding experiments with active learning, we rapidly identify improved compositions and demonstrated up to 3-fold enhancement in protein yield and translation rate for a single reporter gene. We further show that optimization drivers differ between low and high DNA concentrations, revealing that optimal PURE compositions are DNA concentration-dependent. We then apply this optimization strategy to enhance the expression of a 41-kb synthetic chromosome containing 15 genes by maximizing the fluorescence intensities of two reporter proteins. While a 3-fold improvement could be reached on the two gene products guiding learning, a full proteomic analysis revealed that optimization is gene-specific, i.e., changes in PURE system compositions differently impact the amounts of synthesized proteins encoded on the same DNA template. Together, this work establishes active learning as an efficient strategy to navigate the high-dimensional PURE compositional space and provides mechanistic insight into DNA context-dependence of gene expression optimization.

synthetic biology↗

bioRxiv2026

Combinatorial optimization of protein systems in synthetic cells

In vitro reconstitution of protein systems - e.g., metabolic pathways, genetic circuits or biosensors - often requires optimization to enhance their activity. Combinatorial DNA libraries that simultaneously target multiple genes allow for a holistic optimization strategy by studying the interplay between the systems components, which may reveal DNA variants that would be hidden when testing each element in isolation. Here, we screen large populations of synthetic vesicles that express combinatorial DNA variants of a DNA self-replicator or a phospholipid synthesis pathway. We simultaneously vary the strengths of multiple RBSs or synonymously mutate the first codons of multiple genes to explore the effects of the protein translation rates directly on the functionality of the two core synthetic cell modules. We isolated high performers through DNA self-selection or functional screening by fluorescence-activated cell sorting. Long-read sequencing of the fittest variants informed on the optimal RBS strengths and base substitutions in the first codons and indicated which genes were most impactful in regulating the functionality of the protein systems. Single-mutation data were used to predict the fitness of combinatorial variants, which was compared with the experimental fitness observed. The theoretical fitness of combinatorial variants was extremely predictive for the two-gene library of the DNA replicator but less for the larger pathway library. Altogether, our approach exemplifies how combinatorial testing can be expanded from single proteins to multiprotein systems, which can in the future be extended to the evolutionary engineering of even larger genetic and metabolic networks, and eventually an entire artificial cell. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/707944v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7029e4org.highwire.dtl.DTLVardef@1ce778forg.highwire.dtl.DTLVardef@b61b3org.highwire.dtl.DTLVardef@13b6215_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Assembly and cell-free expression of a partial genome for the synthetic cell

The de novo design and assembly of a DNA genome represents an important milestone toward the construction of a minimal synthetic cell. The genome must contain all the instructions to enable primary cellular functions, probably comprising over 150 genes with a total size over 200 kb. Here, we designed and built a partial synthetic genome that satisfies the requirements for its expression in PURE system--a minimal transcription-translation machinery reconstituted from purified elements--and its replication by the protein-primed {varphi}29 DNA replication system. The partial minimal synthetic genome (MSG) has a size of 41 kb, was assembled in yeast from 14 fragments, and harbors genes for phospholipid biosynthesis, DNA replication, and cell division. Yeast marker fragments were added between coding fragments to facilitate screening of correct assemblies and a BAC backbone was included to enable transfer from yeast to E. coli for amplification. Synthesis of all MSG-encoded proteins in PURE system was confirmed by liquid chromatography-mass spectrometry and fluorescence measurements. Moreover, we demonstrate successful compartmentalization and expression of the MSG in liposomes, as well as full-length replication of the linearized MSG by the {varphi}29 DNA replication machinery. This work provides proof-of-concept for the bottom-up assembly and cell-free expression of a functional genome for a minimal synthetic cell.

synthetic biology↗

bioRxiv2025

Image-based phenotypic sorting of synthetic cells

Understanding the relationships between genotype and phenotype is key to many areas of biological research and to the development of synthetic cells. We describe an image-based screening and sorting workflow that explores the phenotypes of gene-expressing vesicles within nonclonal populations and selects the desired variants. Using automated confocal microscopy and real-time, neural-network-assisted image analysis, we demonstrate that liposomes can be selected for fluorescence intensity, protein localization, membrane morphology, and dynamic behaviors, and their phenotype can be linked to genetic content. This approach could substantially accelerate the evolution of cellular functions in a minimal synthetic context.

synthetic biology↗

bioRxiv2025

Autocatalytic selection of gene functions in vitro

The integration of biological functions into a single operating system is considered a major challenge in the construction of a synthetic cell1-3. We present autocatalytic selection (ACS) of gene functions as a driver for integrating biological modules in vitro. A gene of interest (GOI) is introduced into a minimal DNA self-replicator based on the {phi}29 replication machinery4 and the function of the GOI is linked to transcription, translation or DNA replication through a positive feedback loop. As the encoded function eventually promotes DNA self-replication, the gene variants with greater activity are selected. Using different coupling mechanisms, we demonstrate ACS of three functions: transcription, synthesis of a deoxynucleoside triphosphate for DNA replication, and {beta}-galactosidase activity. The latter example illustrates how a function that is unrelated to the Central Dogma can be selected. This work paves the way for ACS-driven Darwinian evolution of virtually any biomolecule in vitro, streamlining the construction of increasingly complex synthetic cells as well as the engineering of biotechnologically relevant enzymes.

synthetic biology↗

bioRxiv2025

Functional selection in a population of synthetic cells with a minimal metabolism

Various synthetic microcompartment systems have been developed to mimic key features of living cells. Here, we focus on artificial cells that capture their capacity to serve as vessels for Darwinian evolution. We assemble micro-compartmentalized In Vitro Transcription-Translation-Replication systems containing a minimal genome, a basic metabolic pathway, a reconstituted protein expression machinery, and a simple DNA replication module, wired in a positive feedback loop. The minimal genome encodes the enzyme deoxyribonucleoside kinase (DNK) whose expression, and then metabolic activity, is required for the genomes replication. We show that these compartments act as minimal Darwinian elements by filtering out non-functional genotypes. We track individual replicators from a library of 42 genetic variants to reveal the systems dynamics at both the population and the single replicator levels. At the population level, we extract the fitness function, which links a genomes metabolic efficiency to its selective success, considering co-encapsulation and hitch-hiking effects. At the individual replicator level, we observe a bimodal distribution of replication yields and propose a mixed model with an inter-droplet heterogeneity with presence or absence of a metabolic feedback loop on the replicator. In addition, we leverage this autonomous self-selection loop to generate a high-resolution mutational map of the DNK enzyme.

synthetic biology↗

bioRxiv2025

A synthetic cell with integrated DNA self-replication and membrane biosynthesis

The emergence, organization, and persistence of cellular life are the result of the functional integration of metabolic and genetic networks. Here, we engineer phospholipid vesicles that can operate three essential functions, namely transcription-translation of a partial genome, self-replication of this DNA program, and membrane synthesis. The synthetic genome encodes six proteins and its compartmentalized expression produces active liposomes with distinct phenotypes demonstrating successful module integration. Our results reveal that genetic factors exert a stronger control over DNA replication and membrane synthesis than metabolic crosstalk or module co-activity. By showing how genetically encoded functions derived from different species can be integrated in liposome compartments, our work opens new avenues for the construction of autonomous and evolving synthetic cells.

synthetic biology↗

bioRxiv2024

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template - encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage - expressed in the protein synthesis using recombinant elements (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

synthetic biology↗

bioRxiv2024

MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification

The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial plasmid libraries. This quick protocol employs the efficient single-stranded DNA annealing protein (SSAP) CspRecT to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in E. coli. In addition to up to 90% single-target plasmid editing efficiency, we demonstrate that MOSAIC enables the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. Lastly, we integrated a user-friendly validation pipeline using Nanopore sequencing reads, requiring minimal computational experience. We anticipate that MOSAIC will provide researchers with a simple, rapid and resource-effective method to edit plasmids or generate large, diverse plasmid libraries for a wide range of in vivo or in vitro applications in molecular and synthetic biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/586135v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@929537org.highwire.dtl.DTLVardef@1825039org.highwire.dtl.DTLVardef@60e89aorg.highwire.dtl.DTLVardef@46ef46_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

synthetic biology↗

bioRxiv2022

Clonal amplification-enhanced gene expression for cell-free directed evolution

In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal {Phi}29 replication machinery and in situ transcription-translation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library either via a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.

synthetic biology↗

bioRxiv2021

Min waves without MinC can pattern FtsA-FtsZ filaments on model membranes

Although the essential proteins that drive bacterial cytokinesis have been identified and reconstituted in vitro, the precise mechanisms by which they dynamically interact to enable symmetrical division are largely unknown. In Escherichia coli, cell division begins with the formation of a proto-ring composed of FtsZ and its membrane-tethering proteins FtsA and ZipA. In the broadly proposed molecular scenario for ring positioning, Min waves composed of MinD and MinE distribute the FtsZ-polymerization inhibitor MinC away from mid-cell, where the Z-ring can form. Therefore, MinC is believed to be an essential element connecting the Min and FtsZ systems. Here, by using cell-free gene expression on planar lipid membranes, we demonstrate that MinDE drive the formation of dynamic, antiphase patterns of FtsZ-FtsA co-filaments even in the absence of MinC. This behavior is also observed when the proteins are compartmentalized inside microdroplets. These results suggest that Z-ring positioning may be achieved with a more minimal set of proteins than previously envisaged, providing a fresh perspective about the role of MinC. Moreover, we propose that MinDE oscillations may constitute the minimal localization mechanism of an FtsA-FtsZ constricting ring in a prospective synthetic cell.

synthetic biology↗

bioRxiv2021

Shaping liposomes by cell-free expressed bacterial microtubules

Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.

synthetic biology↗

bioRxiv2021

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

synthetic biology↗

bioRxiv2020

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. A key event in the bacterial cell cycle is the assembly of cytoskeletal proteins into a ring that defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the membrane and generate budding vesicles, a phenotype that has not been reported before. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results provide new insights on the self-organization of basic cytoskeletal elements involved in bacterial division. Moreover, they demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

synthetic biology↗

bioRxiv2020

Genetically controlled membrane synthesis in liposomes

Lipid membrane, nucleic acids, proteins, and metabolism are essential ingredients for life. Synthetic cellular systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of specific genes activity combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward more effective liposome growth and self-reproduction.

synthetic biology↗

bioRxiv2019

Development of a lipid-based delivery system for the chemotherapeutic compound SN-38

SN-38 is a chemotherapeutic compound with potent antitumor effects. However, its clinical application is currently limited due to its poor solubility and low stability at physiological pH. Liposomes and cyclodextrins have been long studied for the solubilization and delivery of hydrophobic compounds. Aiming to combine the advantages from both systems, we attempted to develop an SN-38-in-cyclodextrin-in-liposome formulation. We found that the encapsulation of SN-38-SBE-{beta}-CD inclusion complexes in the lumen of liposomes was not possible, owing to the disassembly of liposomes and the formation of lipid nanoparticles, as revealed by size exclusion chromatography and single nanoparticle fluorescence microscopy. Interestingly, the retention time of SN-38 inside SN-38-SBE-{beta}-CD-lipid nanoparticles is higher than in liposomes, whereby SN-38 was directly loaded into the lipid film. The toxicity of purified SN-38-SBE-{beta}-CD- lipid nanoparticles was assayed in cultured cancer cells, showing no therapeutic advantage compared to bulk SN-38-SBE-{beta}-CD complexes. Further formulation optimization, in particular an increased concentration of the nanoparticles, will be necessary to obtain cytotoxicity effects. Moreover, the results highlight the value of fluorescence imaging of single, surface-immobilized nanoparticles, in the development of liposomal delivery systems such as drug-in-cyclodextrin- in-liposomes.

biochemistry↗