Vincent Noireaux

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 takes only 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 protocols. We first characterized the lipid interactions of the mechanosensitive channel MscL, then screened 169 Escherichia 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 multienzyme membrane biochemistry.

Materials that change shape or flow typically rely on external fields, photochemical cues or compositions fixed at assembly. Here we introduce a self-morphing active material whose mechanical behaviour is genetically programmed and executed autonomously. By embedding cell-free gene expression within a dense microtubule network, DNA instructions synthesize molecular motors, cross-linkers and transcription factors that continuously generate and regulate active stresses. This experimental platform enables gene-circuit architectures to directly program active-matter dynamics, producing steady, pulsed, arrested and oscillatory mechanical states from genetically encoded biochemical regulation alone. A coarse-grained reaction-mechanics model quantitatively captures these behaviours and shows how gene-circuit parameters, including expression rates, degradation kinetics and regulatory topology, govern the emergence and temporal evolution of active-matter states. Together, these results establish gene circuits as programmable control layers for active matter, providing a general strategy for engineering genetically encoded mechanochemical materials and autonomous active systems.

Bacteriophages are increasingly explored and applied to combat antibiotic-resistant bacteria, yet rapid resistance development and narrow host range can limit their broader application. Engineering phage receptor-binding proteins (RBPs) to expand phage host ranges offers a promising strategy to overcome some of these constraints. However, current approaches remain labor-intensive and difficult to control. Here, we use a cell-free phage engineering workflow to rapidly modify and characterize the RBP of the Salmonella phage LPST153. Using the PHEIGES platform and engineering targeted RBP mutations, we identified residues 389 and 402 within the tail fiber as determinants of host specificity. The G389R substitution significantly increased phage adsorption (%), reaching 65% on E. coli BW25113 and 51% on the LPS-truncated E. coli BW25113 ΔwaaC, compared with 27% and 5.7% for the wild-type LPST153, respectively. Targeted engineering of tail fiber residue 389 generated phage variants with distinct host-range phenotypes, with a G389T variant that exhibited an alternative infection profile relative to G389R. Together, these findings demonstrate that minimal, site-specific modifications of RBPs can substantially modulate LPST153 host specificity. This work establishes a framework for rapid engineering of phages with tailored host ranges, advancing phage-based antimicrobial strategies.

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.

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 RBC’s 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.

Cell-free gene expression (CFE) systems provide a rapid and modular platform for synthesizing bacteriophages without the need for living host cells. However, the generalizability of CFE-based phage synthesis across diverse phages, as well as the functional comparability of in vitro-synthesized phages to host-derived counterparts, has not yet been fully explored. In this study, we evaluated the ability of an Escherichia coli-based CFE platform to synthesize diverse bacteriophages relevant to food safety and synthetic biology applications. Using an E. coli-based CFE system, we synthesized seven phages from four different families, achieving infectious titers ranging from 10⁶ to 10¹¹ plaque-forming units per milliliter (PFU/mL). Of these seven phages, five phages, vB_SalM-LPST153 (LPST153), vB_SenM-S16 (S16), SP6, vB_Sens_Jbel (Jbel), and vB_EcoM_Alf5 (Alf5), are reported here for the first time as successfully synthesized and characterized in a CFE system. CFE-synthesized phages remained capable of infecting host strains; however, significant differences in efficiency of plating (EOP) were observed across multiple Salmonella, E. coli, and Shigella hosts. Long-term storage tests for LPST153 showed that phage buffer (PB) stably maintained CFE titers above 10⁹ PFU/mL for 12 weeks, while purified CFE-synthesized and host-derived LPST153 lysates exhibited broadly comparable thermal stability profiles across the tested temperatures. Using the PHEIGES workflow, we also demonstrated complete in vitro assembly and expression of LPST153 from PCR fragments. In addition, phages were successfully synthesized using CFE lysates derived from the endotoxin-free E. coli strain ClearColi™, demonstrating the synthesis of infectious phages in an endotoxin-free system. These findings expand the diversity of bacteriophages compatible with cell-free synthesis (CFS) and demonstrate that CFE-synthesized phages remain infectious and retain several functional properties, although some differences in host-dependent infectivity were observed. The ability to synthesize infectious phages in endotoxin-free systems and assemble phage genomes entirely in vitro highlights the potential of CFE platforms for scalable, decentralized, and cold-chain-independent manufacturing of phage-based therapeutics and food safety interventions.

Materials that change shape or flow typically rely on external fields, photochemical cues, or compositions fixed at the time of assembly. Here, we introduce a self-morphing active material whose mechanical behavior is programmed genetically and executed autonomously. By embedding cell-free gene expression within a dense microtubule network, DNA instructions are transcribed and translated in vitro into molecular motors, crosslinkers, and transcription factors that generate and regulate internal stresses. We implement two control architectures. In the first, mechanical feedback through co-expression of stress-responsive microtubule crosslinkers produces pulsed dynamics and arrest. In the second, genetic feedback uses motors functionalized with transcription activators to couple mechanical transport to gene regulation, generating sustained oscillations transduced into rhythmic material motion. The system autonomously selects among static, flowing, pulsed, oscillatory, or arrested states, with timescales and amplitudes set by transcriptional programs rather than external fields. These results establish DNA-encoded regulation of active stresses as a general strategy for designing cytoskeletal materials whose mechanical properties are programmed through gene expression.

Plant cytochrome P450 enzymes are central to natural product biosynthesis, but remain difficult to express in microbial hosts due to their transmembrane nature. Lysate-based, cell-free expression systems allow supplementation with artificial membranes to support the expression and translocation of transmembrane proteins. We developed a framework to systematically test liposomal membrane compositions to enhance the plant P450 expression yield. Adjustments to common phospholipid ratios or the addition of plant galactolipids had minimal impact on expression. In contrast, blended liposomes containing Egg PC, sterol-conjugated phospholipids, and PEGylated lipids produced concentration-dependent increases in expression. Expression of an Escherichia coli mechanosensitive channel and three plant P450s improved more than 2-fold, with some P450s showing up to 14-fold enhancement. These findings highlight membrane composition as a key determinant of the P450 expression yield in cell-free expression systems. While P450 activity was not measured, these findings provide a framework for future workflows toward achieving functional plant transmembrane enzymes for the bioproduction of natural products.

Living cells dynamically coordinate gene expression with the spatial organization of their membranes and cytoskeletons. Unraveling how membrane-less organelles arise and interact with cytoskeletal networks remains a fundamental challenge in cell biology, biophysics, and bioengineering. Here, we present a synthetic biology platform that integrates cell-free transcription-translation (TXTL) reactions with phase-separated protein condensates, enabling precise investigation of their interplay with cytoskeletal elements. We show that the bacterial actin homologue MreB forms a scaffold that promotes the wetting of protein condensates in crowded solutions, triggering capillary-driven contraction of the cytoskeletal network. Remarkably, MreB filaments self-assemble into a cortical layer at water-oil droplet interfaces, where their contractile activity leads to wrinkling and global deformation of the synthetic compartment. Our results provide physical insights into how biomolecular condensates and cytoskeletal filaments cooperate to remodel cell-like structures, and establish a minimal model for studying wetting-mediated cytoskeletal dynamics at synthetic membranes.

Phage Felix O1 (FO1) is one of the most widely used bacteriophages for targeting Salmonella enterica and is applied in both research and food safety settings. Despite its broad host range, we observed significantly reduced efficiency of plating (EOP) on a subset of Salmonella enterica strains, including serovar Typhimurium 4/74 ( S . 4/74). We found that O-antigen–mediated steric hindrance contributes to this resistance, which can be partially overcome by supplementation with the SP6 phage tailspike protein (TSP). However, full restoration of the EOP required deletion of the Gifsy-1 prophage, leading to the identification of a single prophage-encoded defense gene, gipd474 (Gifsy-encoded phage defense protein from strain S . 4/74). Heterologous expression of this gene in Escherichia coli increased resistance to multiple tailed phages. Bioinformatic analysis revealed that this gene is widely spread across the Enterobacteriaceae , suggesting a conserved role in phage resistance. Together, these findings uncover a dual barrier to FO1 infectivity in S . 4/74 and highlight gipd474 as a novel, prophage-encoded phage defense gene. IMPORTANCE: Phage therapy and biocontrol are increasingly explored as alternatives to antibiotics, particularly against drug-resistant pathogens such as Salmonella . FO1 is a widely used lytic phage with broad activity across Salmonella serotypes. However, variability in phage infectivity limits its reliability against certain strains. Here, we report that S . 4/74 restricts FO1 through a combination of O-antigen–associated interference and a novel prophage-encoded resistance gene. This gene also impairs the infectivity of other tailed phages and is conserved across multiple Enterobacteriaceae genera. Our findings highlight the importance of prophage-encoded defense systems in shaping phage susceptibility and highlight the need to account for such elements when developing phage-based applications.