Biology subjects

Bonnet, J.

Publications and source records attributed to Bonnet, J..

At least 19 recordsLinked to original sources

bioRxiv2026

The Scm FCS finger binds the Pcl Tudor domain to link canonical PRC1 and PRC2.1 in Drosophila

Canonical Polycomb Repressive Complex 1 (PRC1) was previously reported to selectively associate with Polycomb Repressive Complex 2.1 (PRC2.1) in cross-linking-based affinity purification experiments from Drosophila embryos, but the molecular basis of this interaction has remained unresolved. Here, we established an affinity-purification strategy using cryo-milled Drosophila embryos that recovers intact canonical PRC1 together with the complete PRC2.1 complex without chemical cross-linking. Using AlphaFold3 to systematically model interactions between canonical PRC1 and PRC2.1 subunits, we identified a single high-confidence interface between the FCS finger of the canonical PRC1 accessory subunit Sex comb on midleg (Scm) and the atypical Tudor domain of the PRC2.1 accessory subunit Polycomblike (Pcl). Recombinant Scm FCS finger and Pcl Tudor domain formed a stable complex in vitro, as demonstrated by size-exclusion chromatography and native mass spectrometry. Structure-guided mutagenesis validated the predicted interface: point mutations in either interaction partner abolished complex formation, whereas complementary charge-reversal mutations restored binding. These findings identify the Scm-Pcl interaction as the molecular basis for the selective association between canonical PRC1 and PRC2.1 in Drosophila and reveal that communication between these complexes is mediated by a direct interaction between these accessory subunits.

biochemistry↗

bioRxiv2026

Enemies, more than sex, shape butterfly post-mating odor evolution

Not all odors influencing mating behavior evolve as sex pheromones. Female butterflies post-mating odors have been considered species-specific anti-aphrodisiac pheromones shaped by sexual selection but may also serve broader ecological roles shaped by natural selection. Males transfer odors to females that repel rivals, yet the widespread use of these compounds across phyla makes them targets for eavesdropping, such as by phoretic egg parasitoids. We show that in cabbage white butterflies (Pieris spp.), these odors are highly variable and attract parasitoids, deter predators, and influence oviposition. Using gas chromatography and electroantennography, we demonstrate that odor emission and perception lack species-specificity: compounds once thought unique to P. brassicae and P. rapae are shared across Pieridae. In P. napi, odor variation among populations correlates with parasitoid pressure, but not with latitude, genetic distance, or mating frequency, suggesting ecological rather than sexual drivers. In P. brassicae, CRISPR/Cas9 disruption of odor perception alters oviposition and increases susceptibility to parasitism. Moreover, these odors render females unpalatable to birds. Together, our results show that post-mating odors in Pieris butterflies may act as aposematic signals. We provide evidence that these signals evolve under multiple selective pressures, balancing deterrence of mates and predators, parasitoid avoidance, and host-plant interactions. These findings suggest that chemical signals should be viewed as integrating ecological and reproductive pressures, rather than being interpreted solely through the lens of sexual communication.

ecology↗

bioRxiv2025

A scalable architecture for tuning multistate differentiation ratios in synthetic microbial consortia

Establishing synthetic microbial consortia in competitive environments is often compromised by stochastic colonization bottlenecks, where founder effects lead to the unpredictable dominance of a single strain. Here, we overcome this challenge by engineering a differentiation abacus, a scalable, single-layer recombinase architecture that enables a single progenitor cell to differentiate into up to twelve distinct subpopulations. By arranging competitive excision sites in a linear array, we demonstrate that differentiation ratios can be programmed through rationally tuning recombination-site kinetics and inter-site spacing. This architecture allows the generation of strictly mutually exclusive phenotypes with tunable composition, scaling from simple two-state systems to complex multi-state ensembles without the need for multilayered regulation. Finally, we validate the systems utility in a mouse tumor model, showing that in situ differentiation establishes robust, homogeneous consortia that overcome the colonization variability associated with pre-assembled mixtures. This work provides a versatile and scalable framework for reliably controlling consortia composition for bioproduction, synthetic ecology, and engineered living therapies.

synthetic biology↗

bioRxiv2025

CF2H: a Cell-Free Two-Hybrid platform for rapid protein binder screening

Protein binders that detect, activate, inhibit or otherwise modulate their targets are pivotal for biomedical applications. With the increasing accuracy and accessibility of de novo protein design, faster and cheaper experimental screening methods would democratize and accelerate the identification of high-affinity binders. Here we present Cell-Free Two-Hybrid (CF2H), a rapid and sensitive method for detecting high-affinity protein-protein interactions (PPI) that does not require cloning, protein purification nor high-end laboratory equipment. CF2H uses a dimerization-activated DNA binding domain (DBD) fused to prey and bait proteins to trigger transcription upon protein-protein interaction. We demonstrate that CF2H enables the detection of interactions between various types of target and binder proteins such as single-domain antibodies, DARPins and de novo designed binders. We benchmark CF2H as a screening platform by validating previously reported binders for Mdm2 and discover high-affinity binders targeting the checkpoint inhibitor PD-L1 in less than 24 hours. Finally, we show that CF2H can be used to characterize small-molecules modulators of PPI and detect protein biomarkers, opening the door for a new class of cell-free biosensors.

synthetic biology↗

bioRxiv2024

T cell mediated impairment of epithelial integrity in Crohn's disease

T lymphocytes play a major role in intestinal homeostasis, with a particular impact on the balance between self-renewal and differentiation of intestinal epithelial cells (IECs). In Crohns disease (CD) patients, the intestinal mucosa is inflamed, epithelium permeability is increased, and IECs present compositional and functional defects. The role of T lymphocytes interactions with IECs in the physiopathology of CD remains in question. Here, we use a three-dimensional human autologous coculture model between purified intestinal organoids derived from primary tissues of CD and non-inflammatory control patients, and mucosal T lymphocytes extracted from the same location. We show that while in homeostatic context T cells support the proliferation and differentiation balance of organoids, mucosal T lymphocytes from CD patients present a high cytotoxicity against IECs. Importantly, this cytotoxicity is a persistent defect overtime in culture. Organoids also show defective intestinal stem cells (ISCs) proliferation and morphological changes. Single cell RNA sequencing after coculture highlights a general response of T cells to the epithelial microenvironment, and more particularly, an increase activation of a pro-inflammatory CD8+ T cells effector population in CD patients compared to controls. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/621219v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@78453aorg.highwire.dtl.DTLVardef@8f484org.highwire.dtl.DTLVardef@1ed0934org.highwire.dtl.DTLVardef@f75094_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Fertility Preservation of Vacuum-Dried Ram Spermatozoa Stored for Four Years at Room Temperature

Dry storage at room temperature (RT) could simplify spermatozoa banking. Here, we explored DNA stability and in vitro and in vivo development of embryos derived from vacuum-dried encapsulated (VDE) ram spermatozoa stored for four years or after accelerated aging. While some genomic damage was detected at time 0, DNA fragmentation increased from 3.32{+/-}3% (time 0) to 37.64{+/-}4% (4 years). A decrease in blastocyst rate was observed after four years of storage and 6.7 years of simulated storage (10.2% and 9% versus 13.16% at time 0). Embryo quality, assessed based on Cdx2 and Inf-{tau} gene expression, declined over time. Only two of the 23 embryos transferred into synchronized ewes were implanted but were lost by day 40. In conclusion, dry spermatozoa generated blastocysts after four years of RT storage, but their post-implantation development was impaired. Optimization of the water extraction and storage conditions could better preserve the spermatozoas DNA integrity, resulting in improved embryo quality, compatible with development to term.

physiology↗

bioRxiv2024

The Roboscope: Smart and Fast Microscopy for Generic Event-Driven Acquisition

Automation of fluorescence microscopy is a challenge for capturing rare or transient events in biology and medicine. It relies on smart devices that integrate and interpret the observed data, and react to the targeted biological event. We report on the Roboscope, a novel autonomous microscope combining sequence interruption and deep learning integration, allowing generic event-driven acquisitions. This system distinguishes itself by its adaptability to various experiments, quick capture of dynamic events, and minimal data greediness - training with less than 100 images per class. The Roboscopes capability is demonstrated in non-synchronized cells by capturing the metaphase, a 20-minute event happening once per day or less. Conversely, double thymidine-block synchronisation, despite occurring during DNA replication, may perturb mitotic-spindle mechanics. The Roboscopes versatility and efficiency offer significant advancements to tackle the current challenges of cell biology, spreading out advanced microscopy methods to fundamental research as well as high content screening and precision medicine.

bioengineering↗

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↗

bioRxiv2023

Reference materials for SARS-CoV-2 molecular diagnostic: validation of encapsulated synthetic RNAs for room temperature storage and shipping

The Coronavirus pandemic unveiled the unprecedented need for diagnostic tests to rapidly detect the presence of pathogens in the population. Real-time RT-PCR and other nucleic acid amplification techniques are accurate and sensitive molecular techniques that necessitate positive controls. To meet this need, Twist Bioscience has developed and released synthetic RNA controls. However, RNA is an inherently unstable molecule needing cold storage, costly shipping, and resource-intensive logistics. Imagene provides a solution to this problem by encapsulating dehydrated RNA inside metallic capsules filled with anhydrous argon, allowing room temperature and eco-friendly storage and shipping. Here, RNA controls produced by Twist were encapsulated (RNAshells) and distributed to several laboratories that used them for COVID-19 detection tests by amplification. One RT-LAMP procedure, four different RT-PCR devices and 6 different PCR kits were used. The amplification targets were genes E, N; RdRp, Sarbeco-E and Orf1a/b. RNA retrieval was satisfactory, and the detection was reproducible. RNA stability was checked by accelerated aging. The results for a 10-year equivalent storage time at 25 {degrees}C were not significantly different from those for unaged samples. This room temperature RNA stability allows the preparation and distribution of large strategic batches which can be stored for a long time and used for standardization processes between detection sites. Moreover, it makes it also possible to use these controls for single use and in the field where large temperature differences can occur. Consequently, this type of encapsulated RNA controls, processed at room temperature, can be used as reference materials for the SARS-Cov-2 virus as well as for other pathogens detection.

molecular biology↗

bioRxiv2023

LactoSpanks: a collection of IPTG inducible promoters for the commensal lactic acid bacteria Lactobacillus gasseri.

Lactic acid bacteria (LAB) are important for many biotechnological applications, such as bioproduction and engineered probiotics for therapy. Inducible promoters are key gene expression control elements, yet those available in LAB are mainly based on bacteriocin systems and have many drawbacks, including large gene clusters, costly inducer peptides and little portability to in vivo settings. Using Lactobacillus gasseri, a model commensal bacteria from the human gut, we report the engineering of Lactospanks promoters (Pls), a collection of variable strength inducible promoters controlled by the LacI repressor from B. subtilis and induced by isopropyl {beta}-D-1-thiogalactopyranoside (IPTG). We first show that the Phyper-spank promoter from Bacillus subtilis is functional in L. gasseri, albeit with substantial leakage. We then construct and screen a semi-rational library of Phyper-spank variants to select a set of four IPTG-inducible promoters that span a range of expression levels and exhibit reduced leakages and operational dynamic ranges (from ca. 9 to 28 fold-change). With their low genetic footprint and simplicity of use, Lactospanks will support many applications in L. gasseri, and potentially other lactic acid and gram-positive bacteria.

synthetic biology↗

bioRxiv2022

An Optimized Electrotransformation Protocol For Lactobacillus Jensenii.

Engineered bacteria are promising candidates for in situ detection and treatment of diseases. The female uro-genital tract presents several pathologies, such as sexually transmitted diseases or genital cancer, that could benefit from such technology. While bacteria from the gut microbiome are increasingly engineered, the use of chassis isolated from the female uro-genital resident flora has been limited. A major hurdle to implement the experimental throughput required for efficient engineering in these non-model bacteria is their low transformability. Here we report an optimized electrotransformation protocol for Lactobacillus jensenii, one the most widespread species across vaginal microflora. Starting from classical conditions, we optimized buffers, electric field parameters, cuvette type and DNA quantity to achieve an 80-fold improvement in transformation efficiency, with up to 3.5{middle dot}103 CFUs/{micro}g of DNA in L. jensenii ATCC 25258. We also identify several plasmids that are maintained and support reporter gene expression in L. jensenii. Finally, we demonstrate that our protocol provides increased transformability in three independent clinical isolates of L. jensenii. This work will facilitate the genetic engineering of L. jensenii and enable its use for addressing challenges in gynecological healthcare.

microbiology↗

bioRxiv2022

Evaluating and mitigating clinical samples matrix effects on TX-TL cell-free performance

Cell-free biosensors are promising tools for medical diagnostics, yet their performance can be affected by matrix effects arising from the sample itself or from external components. Here we systematically evaluate the performance and robustness of cell-free systems in serum, plasma, urine, and saliva using two reporter systems, sGFP and luciferase. In all cases, clinical samples have a strong inhibitory effect. Of different inhibitors, only the RNase inhibitor mitigated matrix effects. However, we found that the recovery potential of RNase inhibitor was partially muted by interference from glycerol contained in the commercial buffer. We solved this issue by designing a strain producing an RNase inhibitor protein requiring no additional step in extract preparation. Furthermore, our new extract yielded higher reporter levels than previous conditions and tempered interpatient variability associated with matrix effects. This systematic evaluation and improvements of cell-free system robustness unified across many types of clinical samples is a significant step towards developing cell-free diagnostics for a wide range of conditions.

synthetic biology↗

bioRxiv2022

PeroxiHUB: a modular cell-free biosensing platform using H2O2 as signal integrator

Cell-free systems have great potential for delivering robust, cheap, and field-deployable biosensors. Many cell-free biosensors rely on transcription factors responding to small molecules, but their discovery and implementation still remain challenging. Here we report the engineering of PeroxiHUB, an optimized H2O2-centered sensing platform supporting cell-free detection of different metabolites. H2O2 is a central metabolite and a by-product of numerous enzymatic reactions. PeroxiHUB uses enzymatic transducers to convert metabolites of interest into H2O2, enabling rapid reprogramming of sensor specificity using alternative transducers. We first screen several transcription factors and optimize OxyR for the transcriptional response to H2O2 in cell-free, highlighting the need for pre-incubation steps to obtain suitable signal-to-noise ratios. We then demonstrate modular detection of metabolites of clinical interest - lactate, sarcosine, and choline - using different transducers mined via a custom retro-synthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the pre-incubation step is crucial for optimal sensor operation. Finally, we show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Given the wide range of enzymatic reactions producing H2O2, the PeroxiHUB platform will support cell-free detection of a large number of metabolites in a modular and scalable fashion.

synthetic biology↗

bioRxiv2022

Rapid prototyping of metabolites detection by bacterial biosensors in human fecal samples.

Gut metabolites are pivotal mediators of host-microbiome interactions and provide an important window on human physiology and disease. However, current methods to monitor gut metabolites rely on heavy and expensive technologies such as liquid chromatography-mass spectrometry (LCMS). In that context, robust, fast, field-deployable, and cost-effective strategies for monitoring fecal metabolites would support large-scale functional studies and routine monitoring of metabolites biomarkers associated with pathological conditions. Living cells are an attractive option to engineer biosensors due to their ability to detect and process many environmental signals and their self-replicating nature. Here we optimized a protocol for feces processing and gut metabolites detection using bacterial biosensors (bactosensors), enabling rapid evaluation of their operational capacity in these samples. We show that a simple filtration step is enough to remove host microbes and reproducibly obtain a physiological-derived media retaining important characteristics of human feces, such as matrix effects and endogenous metabolites. We measured how fecal samples affect the performance of biosensors for benzoate, lactate, anhydrotetracycline, and bile acids, and found that bactosensors are highly sensitive to fecal matrices. Sensitivity to the matrix is biosensor-dependent but also varies between individuals, highlighting the need for case-by-case optimization for bactosensors operation in feces. Finally, by detecting endogenous bile acids, we demonstrate that bactosensor can be used for metabolites monitoring in feces. This work lays the foundation for the optimization and use of bacterial biosensors for fecal metabolites monitoring. In the future, our method could also allow rapid pre-prototyping of engineered bacteria designed to operate in the gut, with applications to in situ diagnostics and therapeutics.

synthetic biology↗

bioRxiv2021

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from {sigma}70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the {Delta}recBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

synthetic biology↗

bioRxiv2021

Long term conservation of DNA at ambient temperature. Implications for DNA data storage

DNA conservation is central to many applications. This leads to an ever-increasing number of samples which are more and more difficult and costly to store or transport. A way to alleviate this problem is to develop procedures for storing samples at room temperature while maintaining their stability. A variety of commercial systems have been proposed but they fail to completely protect DNA from deleterious factors, mainly water. On the other side, Imagene company has developed a procedure for long-term conservation of biospecimen at room temperature based on the confinement of the samples under an anhydrous and anoxic atmosphere maintained inside hermetic capsules. The procedure has been validated by us and others for purified RNA, and DNA in buffy coat or white blood cells lysates, but a precise determination of purified DNA stability is still lacking. We used the Arrhenius law to determine the DNA degradation rate at room temperature. We found that extrapolation to 25 {degrees}C gave a degradation rate constant equivalent to about 1 cut/century/100 000 nucleotides, a stability several orders of magnitude larger than the current commercialized processes. Such a stability is fundamental for many applications such as the preservation of very large DNA molecules (particularly interesting in the context of genome sequencing) or oligonucleotides for DNA data storage. Capsules are also well suited for this latter application because of their high capacity. One can calculate that the 64 zettabytes of data produced in 2020 could be stored, standalone, for centuries, in about 20 kg of capsules.

biochemistry↗

bioRxiv2021

A synthetic receptor platform enables rapid and portable monitoring of liver dysfunction via engineered bacteria.

Bacterial biosensors, or bactosensors, are promising field-deployable agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we present a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We applied EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improved the sensitivity and lowered the limit-of-detection of the sensing module by directed evolution. We then engineered a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

synthetic biology↗

bioRxiv2021

An engineered lactate responding promoter system operating in glucose-rich and anaerobic environments.

Bacteria equipped with genetically-encoded lactate biosensors would support several applications in biopharmaceutical production, diagnosis, or therapeutics. However, many applications involve glucose-rich and anaerobic environments, in which current whole-cell lactate biosensors have low performance. Here we engineered a synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expression to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anaerobia) can operate in glucose rich, aerobic and anaerobic conditions. Our work provides a versatile lactate biosensing platform suitable for many environmental conditions.

synthetic biology↗