Biology subjects

Noireaux, V.

Publications and source records attributed to Noireaux, V..

At least 19 recordsLinked to original sources

bioRxiv2026

ORB-TXTL: cell-free expression of membrane proteins on lipid bilayer-coated beads

Membrane proteins achieve a remarkable range of cellular functions, yet their characterization at high throughputs remains difficult with standard reconstitution methods. Here, we develop On-bead Reconstitution into Bilayers via Cell-free Transcription and Translation (ORB-TXTL), a platform that uses compositionally tunable lipid bilayer-coated silica beads as scaffolds for cell-free synthesized interacting and integral membrane proteins. ORB-TXTL is fast as it just takes a few hours to integrate membrane proteins onto the beads, which can be extensively washed and seamlessly transferred between reaction buffers, to perform assays that are read out by standard laboratory equipment without tagging and sophisticated equipment. We first characterized the lipid interactions of the mechanosensitive channel MscL, then screened 169 E. coli proteins and identified a systematic dependence of membrane integration efficiency on the number of transmembrane domains. Finally, we functionally reconstituted the E. coli phospholipid synthesis pathway, demonstrating that ORB-TXTL is a tractable and cheap chassis for multi-enzyme membrane biochemistry.

synthetic biology↗

bioRxiv2026

Engineering erythrocytes into synthetic cells using cell-free gene expression

Erythrocytes, commonly known as red blood cells (RBCs), constitute the most abundant cell type in vertebrate mammals. Due to their unique biological and physical attributes, RBCs have been the focus of extensive research in biomedical engineering. Methods have been developed to transform RBCs into adaptable carriers for molecular payloads, thereby extending their functional capabilities beyond what they naturally transport and accomplish. Concurrently with RBCs applied science, cell-free gene expression (CFE) has advanced into a tractable technology that can be integrated with a broad range of materials. In this work, we harness the advantages of CFE to engineer RBCs into hybrid synthetic cells. We encapsulate CFE reactions within RBC ghosts to execute elementary gene circuits, including biosensors, and to synthesize phages from their genomes. Furthermore, we engineer and functionalize the outer membrane of mature RBCs to attach diverse payloads, such as a SARS-CoV-2 antigen recognized by a specific antibody. CFE interfaces remarkably well with RBCs, enabling their rapid, low-cost transformation into red blood synthetic cells (RBSCs) with potential biomedical and biotechnological applications.

bioengineering↗

bioRxiv2026

Programming Metabolic Dependency in Synthetic Cells Under Resource Scarcity

Engineering synthetic cells where metabolism directly controls gene expression is one of the greatest challenges in synthetic biology. This coupling between metabolic activity and protein synthesis is also thought to have been a vital step in the evolution of the earliest cellular life. This fundamental process is essential for developing minimal, self-sustaining cells, both engineered for biotechnology applications and as models explaining the origins of life. Here, we present a programmable cell-free platform that links metabolic activity to translation under defined resource limitations. Using an engineered amino-acid-dependent cell-free translation system, we introduced a tunable metabolic bottleneck (depleting tyrosine). This enabled imposing a controlled metabolic constraint on protein synthesis. To alleviate this constraint, we then incorporated phenylalanine hydroxylase (PAH) as a minimal module for tyrosine synthesis. The PAH-driven tyrosine synthesis established a system in which protein expression is directly controlled by amino acid biosynthesis. This relationship was recapitulated in liposome compartments, resulting in three distinct synthetic cell populations. The metabolically active population had significantly higher fitness in protein production. Overall, this work establishes an experimentally tractable platform to investigate how primitive cells may have evolved internal metabolic capabilities, and it represents a foundational step toward constructing more autonomous and self-regulating synthetic cells.

biochemistry↗

bioRxiv2025

A synthetic cell phage cycle

Viral infection of living cells, exemplified by bacteriophage interaction with bacteria, is fundamental to biology and universal across living systems. Here, we establish an all-cell-free viral cycle where T7 phages infect synthetic cells, equipped with lipopolysaccharides on the outer leaflet of the lipid membrane while encapsulating a cell-free gene expression system. We track each cycle step to demonstrate T7 phage-specific adsorption onto the liposomes, genome ejection, replication, expression, and assembly of new infectious virions within the synthetic cells. We quantify key characteristics of the cycle, including the multiplicity of infection, replication efficiency, liposome size constraints, and phage rebinding dynamics. This work establishes a versatile, fully defined in vitro platform for reconstructing and investigating viral infections from individual molecular components.

synthetic biology↗

bioRxiv2024

Carbon-conserving Bioproduction of Malate in an E. coli-based Cell-Free System

Formate, a biologically accessible form of CO2, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an E. coli-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that in situ regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 M malate after 8 hours. This system conserves 43% of carbon otherwise lost as CO2 and incorporates 0.13 mol CO2 equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals. HighlightsO_LISuccessfully engineered the carbon-conserving reductive TCA and formate assimilation pathways in a lysate-based cell-free system for production of the C4 industrial di-acid malate from C1 and C2 feedstocks. C_LIO_LIAchieved a 6-fold reduction in competition from the endogenous cell-free metabolism by blocking TCA activity using small-molecule inhibitors and lysate dilution. C_LIO_LIIncreased accumulation of malate by 15-fold in a single-step reaction using cell-free expression of an enzymatic cofactor regeneration system. C_LIO_LITechno-economic analysis identified routes for economically feasible production of malate from renewable feedstocks in a cell-free system by improving conversion efficiency and reducing lysate cost. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/623433v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@3c89adorg.highwire.dtl.DTLVardef@72d9b3org.highwire.dtl.DTLVardef@327e2eorg.highwire.dtl.DTLVardef@fa0964_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Cell-free immuno-profiling on a genetically programmed biochip

Emerging cell-free synthetic biology approaches provide biosafe, cheap, and versatile genetic tools to advance therapeutic research and development. Combined with micro-fabrication technology, we developed a platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies and human receptors in miniaturized compartments on a silicon chip. Photolithographic surface patterning of protein traps and on chip expression from high density gene brushes generated a continuous surface density gradient of fluorescently labeled antigens. Antibodies binding to the antigen gradient generate a full binding curve in each single compartment for affinity determination. We used the SARS-CoV-2 antigens as a model to profile the specificity and affinity of monoclonal antibodies to > 30 viral epitopes synthesized simultaneously on one chip in a genotype-phenotype linked compartments. We further profiled polyclonal antibodies in minute volumes of human sera, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. Cell-free co-synthesis of the human ACE2 receptor with the viral Receptor-Binding-Domain yielded relative binding affinities to different SARS-CoV-2 variants. This rapid, quantitative, and on-chip genetically programmed approach allows to study complex protein-protein interactions independent of protein purification steps for human immuno-profiling with a fast response time for combating emerging pathogens.

bioengineering↗

bioRxiv2024

Transcription swells chromosomes in vitro

Transplanting chromosomes from living to artificial cells would impact our understanding of chromosome organization and DNA transactions, with implications for autonomous biological systems. Here, we transplanted Escherichia coli chromosomes into artificial cells, enabling real-time labeling, manipulation, and steady-state gene expression down to the single-molecule limit. Chromosomes stripped of native proteins transitioned from a swollen to compacted state induced by transcription inhibition, in contrast to protein-bound chromosomes retaining an organization with blobs. In a cell-free expression reaction, RNA polymerases were uniformly distributed along the entire chromosome and rapidly detached, consistent with a global transcriptional activity. We used tailored surfaces to capture and count 20 nascent proteins per hour from a single gene on the chromosome. We mapped stably bound condensins to the blobs, supporting a model where swelling by transcription is counterbalanced by condensin-mediated compaction. Our data suggest transplanted chromosomes as active gels organized by molecular machines.

biophysics↗

bioRxiv2024

Red Light Mediated Photo-Conversion of Silicon Rhodamines to Oxygen Rhodamines for Single-Molecule Microscopy

The rhodamine motif has been modified in myriad ways to produce probes with specific fluorescent and chemical properties optimal for a variety of microscopy experiments. Recently, far-red emitting silicon rhodamines have become popular in single-molecule localization microscopy (SMLM), since these dyes are membrane-permeable and can be used alongside red fluorophores for two-color imaging. While this has expanded multi-color SMLM imaging capabilities, we demonstrate that silicon rhodamines can create previously unreported photoproducts with significantly blueshifted emissions, which appear as bright single-molecule crosstalk in the red emission channel. We show that this fluorescence is caused by the replacement of the central silicon group with oxygen after 640 nm illumination, turning far-red silicon rhodamines (JFX650, JF669, etc.) into their red oxygen rhodamine counterparts (JFX554, JF571, etc.). While this blueshifted population can cause artifacts in two-color SMLM data, we demonstrate up to 16-fold reduction in crosstalk using oxygen-scavenging systems. We also leverage this far-red photoconversion to demonstrate UV-free photoactivated localization microscopy (PALM) without the need for additives, and with 5-fold higher efficiency than the Cy5 to Cy3 conversion. Finally, we demonstrate multiplexed pseudo two-color PALM in a single emission channel by separating localizations by their photo-activation wavelengths instead of their emission wavelengths. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/595223v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@141fe66org.highwire.dtl.DTLVardef@1a4f9f5org.highwire.dtl.DTLVardef@16605dorg.highwire.dtl.DTLVardef@1109255_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Split minireporters facilitate monitoring of gene expression and peptide production in linear cell-free transcription-translation systems

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present minireporters based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that minireporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP, and matching with in vivo expression measurements. We also use minireporters as small fusion tags to measure TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent minireporter based on split-nanoluciferase. The [~]80% gene sequence length reduction afforded by minireporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of minireporters compared to full-length GFP is less likely to bias peptide solubility assays. We anticipate that minireporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

synthetic biology↗

bioRxiv2024

Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins

Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report TXTL-QCMD to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes or oscillating interactions by the E. coli Min system. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system unamenable to labeling, we discovered that ZorA and ZorB integrate together within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.

biophysics↗

bioRxiv2024

On-chip large-scale-integration and 2D collective modes of genetically programmed artificial cells

The on-chip large-scale-integration of genetically programmed artificial cells capable of exhibiting collective modes is an important goal for fundamental research and technology. Here, we report assembly of a 2D layout of 1024 monolithic DNA compartments as artificial cells on a 5-millimeter square silicon chip. Homeostatic cell-free protein synthesis reactions driven by genetic circuits occur inside the compartments. We created a reaction-diffusion system with a 30x30 square lattice of artificial cells interconnected by thin capillaries for diffusion of products. Driving the system by a genetic oscillator revealed emergent collective modes of synchrony and propagating phase waves in 2D, with dynamics controlled by geometry. This demonstrates a class of nonequilibrium autonomous systems, where chemical energy consumed to make proteins induces 2D collective patterns of gene expression on multicellular scales, with applications in biological computing, sensing, and materials synthesis.

biophysics↗

bioRxiv2023

PHEIGES, all-cell-free phage synthesis and selection from engineered genomes

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

synthetic biology↗

bioRxiv2023

A genetic circuit on a single DNA molecule as autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcriptiontranslation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we used a genetically encoded fluorescent reporter system with improved spatiotemporal resolution and observed the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally designed a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibited enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that coexpressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

synthetic biology↗

bioRxiv2021

Critical role of DNA unwinding and Cas9 conformational changes in modelling off-target effects

CRISPR-Cas9 off-target effects interfere with the ability to accurately perform genetic edits. To predict off-target effects CRISPR-Cas9 researchers perform high throughput guide RNA mismatch and bulge experiments and then use the data to fit thermodynamic binding models. While impactful from an engineering perspective such models are not based on the experimentally observed target interrogation process and thus incorrectly measure the energetic effects mismatches have on the system. In this work we convert an experimentally deduced qualitive model of target interrogation to a linear ODE model and demonstrate that the mismatch tolerance patterns observed in experiments do not need to be caused by differences in energetic penalties of mismatches but rather are emergent effects of the timing and coordination of target DNA unwinding and Cas9 conformational changes.

biophysics↗

bioRxiv2020

Decision-making in a synthetic cell: the limits of biological computation

We measured the dynamics of decision-making by a minimal bistable gene network integrated in a synthetic cell model, free of external perturbations. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow computation to a fuzzy and rapid regime dominated by singleprotein fluctuations. Fuzzy computation appeared at DNA and protein concentrations 100-fold lower than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. Whereas the high-copy regime was characterized by a sharp transition, hysteresis and robust memory, the low-copy limit showed incipient strong fluctuations, switching between states, and a signature of cellular individuality across the decision-making point. Our work establishes synthetic cells operating rapidly at the single molecule level to integrate gene regulatory networks with metabolic pathways for sustained survival with low energetic cost.One Sentence Summary Decision-making in a synthetic cell can be slow and precise or rapid and probabilistic by reducing the number of computing molecules by five decades down to single-molecule fluctuations.Competing Interest StatementThe Noireaux laboratory receives research funds from Arbor Biosciences, a distributor of the myTXTL cell-free protein synthesis kit.View Full Text

synthetic biology↗

bioRxiv2019

In vitro implementation of robust gene regulation in a synthetic biomolecular integral controller

Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties. Motivated by the need to tune the dynamics and improve the robustness of synthetic gene circuits, biological engineers have proposed various designs that mimic natural molecular feedback control mechanisms. However, practical and predictable implementations have proved challenging because of the complexity of synthesis and analysis of complex biomolecular networks. Here, we analyze and experimentally validate a first synthetic biomolecular controller executed in vitro. The controller is based on the interaction between a sigma and an anti-sigma factor, which ensures that gene expression tracks an externally imposed reference level, and achieves this goal even in the presence of disturbances. Our design relies upon an analog of the well-known principle of integral feedback in control theory. We implement the controller in an Escherichia coli cell-free transcription-translation (TXTL) system, a platform that allows rapid prototyping and implementation. Modeling and theory guide experimental implementation of the controller with well-defined operational predictability.

synthetic biology↗

bioRxiv2018

Multiplex transcriptional characterizations across diverse and hybrid bacterial cell-free expression systems

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is often limited by the use of single-channel reporter assays. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employed this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from >1,000 diverse regulatory sequences revealed functional differences in gene expression that could be predictively modeled. Finally, we constructed and examined the transcriptional capacities of dual-species \"hybrid\" cell lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic circuit prototyping in new bacterial chassis for synthetic biology.

synthetic biology↗

bioRxiv2018

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

synthetic biology↗