Biology subjects

Schaerli, Y.

Publications and source records attributed to Schaerli, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Harnessing Escherichia coli motility to engineer bacterial Voronoi patterns

Cell motility drives spatial pattern formation across diverse biological systems. Here, we engineer Escherichia coli motility in semi-solid agar to control Voronoi patterns in two and three dimensions, partitioning space into regions closest to their respective inoculation seeds. Consistent with our reaction-diffusion model, we observed that collisions between expansion fronts generate either biomass depletion (''gaps'') or accumulation (''anti-gaps''), governed by the relative diffusion rates of bacteria and nutrients. By engineering strains with distinct expansion rates and tuneable motility, and by integrating these experimental data into a dynamic Voronoi model, we achieved precise control over pattern geometry. This enabled the generation of gaps with varying widths, curved boundaries, asymmetric structures, seedless regions, and complex composite patterns. Together, these findings establish bacterial Voronoi patterns as a programmable platform for engineering multicellular spatial organization, with potential applications in synthetic biology and materials science.

synthetic biology↗

bioRxiv2026

Competition between co-localized gut symbionts underlies inter-individual variation in the honeybee gut microbiota

Microbes in the animal gut compete to colonize spatially restricted host niches. However, whether competition for space drives inter-individual variation among hosts, and which factors determine the outcome of such competition, remain poorly understood. Here, we investigated competitive interactions of the western honeybee gut symbiont Frischella perrara with other members of the bee gut microbiota. Shotgun metagenomics analysis of individual bees revealed that F. perrara is negatively correlated with a specific species of the genus Gilliamella. Co-colonization of microbiota-depleted bees with these two bacteria resulted in their competitive exclusion. The outcome of this competition depended on the relative number of bacteria each bee received and benefited Gilliamella when one of the two type VI secretion systems of F. perrara was mutated. Using fluorescently tagged strains, high-resolution microscopy, and gut region-specific quantification, we show that both bacteria localize to the same host niche in the ileum of mono-colonized bees, indicating competition in a spatially restricted host niche. Moreover, both microbes protected against infection, promoting bee health. This competition provides an explanatory mechanism underlying variation in the occurrence of F. perrara across honeybee colonies and highlights the importance of gut spatial structure and microbial competition in shaping microbiome composition and inter-individual variability.

microbiology↗

bioRxiv2025

An engineered multi-step differentiation program in Escherichia coli for self-organized spatial patterning

In nature, complex multicellular structures originate from individual cells containing all essential information for differentiation, patterning and morphogenesis. Synthetic biology enables a bottom-up approach to study these processes by engineering and combining individual modules to progressively increase the systems complexity. Here, we engineered a multi-step program mimicking cell differentiation in the model prokaryote Escherichia coli. Starting from genetically identical cells and without providing any external positional information, we generated autonomous spatial patterns of colonies on a solid surface. We first employed a toggle switch to break population homogeneity (symmetry breaking), stochastically differentiating cells into two subpopulations: senders and receivers. Next, we activated expression of a third reporter in receiver colonies located in close proximity to sender colonies via quorum-sensing based communication (paracrine signaling). Finally, we mimic maturation of the newly emerged population by expressing a fourth reporter via an orthogonal, self-activating, quorum sensing signal (autocrine signaling). The diversity of spatial patterns generated by this multi-step program was accurately captured by simulations of a corresponding mathematical model. Together, these results demonstrate that multi-step differentiation programs can be engineered in unicellular bacteria to drive fully self-organized spatial pattern formation.

synthetic biology↗

bioRxiv2025

Engineered symbiont biosensor maps micron-scale sugar gradients in the honeybee gut

The honeybee gut microbiota plays a key role in shaping host health and susceptibility to disease. Yet, the nutrient environment it experiences within the gut remains poorly characterized. In particular, little is known about the spatial distribution of nutrients across this community, as resolving such fine gradients in vivo has been technically challenging. Here, we engineer the native honeybee symbiont Snodgrassella alvi as a living biosensor to quantify the bioavailability of the dietary sugar arabinose within the gut. By expanding the genetic toolkit for S. alvi through chromosomal integration of high-burden genes and a suite of low-strength promoters, we achieve stable multi-gene expression without compromising host colonization. The resulting biosensor generates a specific, dose-dependent response to arabinose in situ, enabling visualization of sugar gradients across gut-associated bacterial biofilms at micron-scale resolution. Using this system, we show that diet-derived arabinose distribution is highly heterogeneous and can be influenced by the metabolic activity of co-colonizing Gilliamella species. These findings highlight how diet composition and microbial specialization generate fine-scale microenvironments within the gut. More broadly, this work further establishes S. alvi as a genetically tractable platform for in situ probing of microbial metabolism and nutrient distribution.

synthetic biology↗

bioRxiv2025

Intracellularly Coupled Oscillators for Synthetic Biology

Synthetic biology aims to engineer or re-engineer living systems. To achieve increasingly complex functionalities, it is beneficial to use higher-level building blocks. In this study, we focus on oscillators as such building blocks, propose novel oscillator-based circuit designs and model the interactions of intracellularly coupled oscillators. We classify these oscillators on the basis of coupling strength: independent, weakly or strongly, and deeply coupled. We predict a range of fascinating dynamic behaviours to arise in these systems, such as the beat phenomenon, amplitude and frequency modulation, period doubling, higher-period oscillations, chaos, resonance, and synchronization, with the aim of guiding future experimental work in bacterial synthetic biology. Finally, we outline potential applications, including oscillator-based computing that integrates processing and memory functions, offering multistate and nonlinear processing capabilities.

synthetic biology↗

bioRxiv2025

Let there be multifunctionality: Uncovering the criticality zoo of the AC-DCgenetic circuit

Gene regulatory networks (GRNs) govern processes such as cell fate, patterning, and adaptation. While multistability and oscillations are both common GRN dynamics in cell biology, they are typically studied and engineered in isolation. Here, we challenge this separation using the AC-DC circuit, a minimal three- gene network that merges the classical toggle switch and repressilator. Using a thermodynamic formalism and Bayesian inference, we show that even a single-inducer version of the circuit can display diverse mul-tifunctional dynamics, including the coexistence of oscillations and multistability. In addition we explore robustness, classify emergent behaviours, and analyse critical slowing down and regime transitions. Re-markably, the AC-DC circuit can produce more than 30 topologically distinct bifurcation diagrams, chal-lenging the classical view that network topology rigidly constrains dynamical outcomes. This flexibility enables synthetic capabilities that couple hysteresis with oscillations, critical slowing down, and reversibility. By uncovering the hidden potential of minimal genetic circuits and outlining design principles for their implementation, this work opens new directions for harnessing emergent complexity using the basic building blocks of life.

systems biology↗

bioRxiv2024

GRN modeler: An Intuitive Tool for Constructing and Evaluating Gene Regulatory Networks and its Applications to Oscillators and a Light Biosensor

Modeling and simulating gene regulatory networks (GRNs) is crucial for understanding biological processes, predicting system behavior, guiding the design of synthetic biological systems, and interpreting experimental data. In synthetic biology, GRNs play a pivotal role in enabling the design and control of complex systems for a wide range of applications. However, GRN simulations can be time-consuming and often require specialized expertise. To make this process more accessible, we developed a user-friendly application with a graphical user interface (GUI), allowing users to create simple phenomenological models without requiring prior programming experience. We demonstrate the versatility of our tool through several examples, including the design of novel oscillator families capable of robust oscillation with an even number of nodes. These complement the well-known repressilator family, which only oscillates with odd-numbered nodes. Furthermore, we showcase how GRN modeler allowed us to develop a light-detecting biosensor in Escherichia coli that can track light intensity over several days, leaving a record in the form of ring patterns in bacterial colonies. In summary, our work empowers biologists to model their systems of interest even without programming expertise.

synthetic biology↗

bioRxiv2024

Engineering Intercellular Communication using M13 Phagemid and CRISPR-based Gene Regulation for Multicellular Computing in Escherichia coli

Engineering multicellular consortia, where information processing is distributed across specialized cell types, offers a promising strategy for implementing sophisticated biocomputing systems. However, a major challenge remains in establishing orthogonal intercellular communication, or "wires", within synthetic bacterial consortia. In this study, we address this bottleneck by integrating phagemid-mediated intercellular communication with CRISPR-based gene regulation for multicellular computing in synthetic E. coli consortia. We achieve intercellular communication by regulating the transfer of single guide RNAs (sgRNAs) encoded on M13 phagemids from sender to receiver cells. Once inside the receiver cells, the transferred sgRNAs mediate gene regulation via CRISPR interference. Leveraging this approach, we successfully constructed one-, two-, and four-input logic gates. Our work expands the toolkit for intercellular communication and paves the way for complex information processing in synthetic microbial consortia, with diverse potential applications, including biocomputing, biosensing, and biomanufacturing.

synthetic biology↗

bioRxiv2024

Engineering microbial consortia: uptake and leakage ratedifferentially shape community arrangement and composition

Bacteria often grow as communities in intricate spatial arrangements on surfaces and interact with each other through the local exchange of diffusible molecules. Yet, our understanding of how these interactions shape the properties of the communities remains limited. Here, we study synthetic communities of Escherichia coli amino acid auxotrophs interacting through the obligate exchange of amino acids. We genetically engineer these strains to alter their amino acid leakage and uptake abilities. We then characterise the spatial arrangement and composition of the communities when grown on a surface. By integrating experimental data with mathematical modeling, we demonstrate that amino acid uptake and leakage rates are crucial determinants of community structure. Our results show that while the spatial arrangement of the community is primarily governed by uptake rates, the community composition is predominantly influenced by leakage rates. These findings enhance our understanding of microbial community dynamics and provide a framework for predicting and engineering microbial consortia.

microbiology↗

bioRxiv2024

From resonance to chaos: modulating spatiotemporal patterns through a synthetic optogenetic oscillator

Oscillations are a recurrent phenomenon in biological systems across scales, including circadian clocks, metabolic oscillations and embryonic genetic oscillators. Despite their fundamental significance in biology, deciphering core principles of biological oscillators is very challenging due to the multiscale complexity of genetic networks and the difficulty in perturbing organisms in vivo. In this study, we tackle this challenge by re-designing the well-characterised synthetic oscillator, known as "repressilator", in Escherichia coli and controlling it using optogenetics, thus introducing the "optoscillator". When we apply periodic light pulses, the optoscillator behaves as a forced oscillator. Bacterial colonies harboring synthetic oscillators manifest oscillations as spatial ring patterns. Leveraging this feature, we systematically investigate the number, intensity and sharpness of the rings under different regimes of light exposure. By integrating experimental approaches with mathematical modeling, we show that this simple oscillatory circuit can generate complex dynamics that, depending on the external periodic forcing, are transformed into distinct spatial patterns. We report the observation of synchronisation, resonance, undertone and period doubling. Furthermore, we present evidence supporting the existence of a chaotic regime. This work highlights the intricate spatiotemporal patterns accessible by synthetic oscillators and underscores the potential of our approach in understanding the underlying principles governing biological oscillations.

synthetic biology↗

bioRxiv2023

Fecal transplant allows transmission of the gut microbiota in honey bees

The gut of honey bees is colonized by symbiotic bacteria during the first days of adult life, once bees have emerged from their wax cells. Within five days, the gut microbiota becomes remarkably stable and consistent across individual bees. Yet, the modes of acquisition and transmission of the gut microbiota are to be confirmed. Few studies suggested bees could be colonized via contact with fecal matter in the hive and via social interactions. However, the composition of the fecal microbiota is still unknown. It is particularly unclear whether all bacterial species can be found viable in the feces and can therefore be transmitted to newborn nestmates. Using 16s rRNA gene amplicon sequencing we revealed that the composition of the honey bee fecal microbiota is strikingly similar to the microbiota of entire guts. We found that fecal transplantation resulted in gut microbial communities largely similar to those obtained from feeding gut homogenates. Our study shows that fecal sampling and transplantation are viable tools for the longitudinal analysis of bacterial community composition and host-microbe interactions. Our results also imply that contact of young bees with fecal matter in the hive is a plausible route for the acquisition of the core gut microbiota.

microbiology↗

bioRxiv2023

A direct experimental test of Ohno's hypothesis

Gene duplication drives evolution by providing raw material for proteins with novel functions. The oldest and historically most influential hypothesis about the evolutionary fate and potential of duplicated genes has been proposed by Susumu Ohno in 1970. This hypothesis essentially posits that gene duplication can help genes tolerate new mutations and thus facilitates the evolution of new phenotypes. Competing hypotheses argue that deleterious mutations will usually inactivate gene duplicates too rapidly for Ohnos hypothesis to work. Here, we provide a first direct experimental test of Ohnos hypothesis. Specifically, we evolved one or exactly two copies of a gene encoding a fluorescent protein in Escherichia coli through multiple rounds of mutagenesis and selection. We then analyzed the genotypic and phenotypic evolutionary dynamics of the evolving populations through high-throughput DNA sequencing, biochemical assays, and engineering of selected variants. In support of Ohnos hypothesis, populations carrying two gene copies displayed higher mutational robustness than those carrying a single gene copy. As a consequence, the double-copy populations experienced relaxed purifying selection, evolved higher phenotypic and genetic diversity, carried more mutations and accumulated combinations of key beneficial mutations earlier. However, their phenotypic evolution was not accelerated, possibly because one gene copy rapidly became inactivated by deleterious mutations. Our work provides an experimental platform to test models of evolution by gene duplication, and it supports alternatives to Ohnos hypothesis that point to the importance of gene dosage.

evolutionary biology↗

bioRxiv2023

Synthetic gene circuits combining CRISPR interference and CRISPR activation in E. coli: importance of equal guide RNA binding affinities to avoid context-dependent effects

Gene expression control based on CRISPR has emerged as a powerful approach for constructing synthetic gene circuits. While the use of CRISPR interference (CRISPRi) is already well-established in prokaryotic circuits, CRISPR activation (CRISPRa) is less mature and combination of the two in the same circuits is only just emerging. Here, we report that combining CRISPRi with SoxS-based CRISPRa in Escherichia coli can lead to context-dependent effects due to different affinities in the formation of CRISPRa and CRISPRi complexes, resulting in loss of predictable behaviour. We show that this effect can be avoided by using the same scaffold guide RNA structure for both complexes.

synthetic biology↗

bioRxiv2023

Rewiring capsule production by CRISPRi-based genetic oscillators demonstrates a functional role of phenotypic variation in pneumococcal-host interactions

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence1-8, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes9-14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15-20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

microbiology↗

bioRxiv2023

Engineering a symbiont as a biosensor for the honey bee gut environment

The honey bee is a powerful model system to probe host-gut microbiota interactions, and an important pollinator species for natural ecosystems and for agriculture. While bacterial biosensors can provide critical insight into the complex interplay occurring between a host and its associated microbiota, the lack of methods to non-invasively sample the gut content, and the limited genetic tools to engineer symbionts, have so far hindered their development in honey bees. Here, we built a versatile molecular toolkit to genetically modify symbionts and report for the first time in the honey bee a technique to sample their feces. We reprogrammed the native bee gut bacterium Snodgrassella alvi as a biosensor for IPTG, with engineered cells that stably colonize the gut of honey bees and report in a dose-dependent manner exposure through the expression of a fluorescent protein. We showed that fluorescence readout can be measured in the gut tissues or non-invasively in the feces. These tools and techniques will enable rapid building of engineered bacteria to answer fundamental questions in host-gut microbiota research.

synthetic biology↗

bioRxiv2022

Environment-dependent epistasis increases phenotypic diversity in gene regulatory networks

Mutations to gene regulatory networks can be maladaptive or a source of evolutionary novelty. Epistasis confounds our understanding of how mutations impact the expression patterns of gene regulatory networks, because such nonlinearities make it difficult to predict the combined phenotypic effects of mutations based on knowledge of the mutations individual effects. This challenge is exacerbated by the dependence of epistasis on the environment, which is particularly germane to gene regulatory networks that interpret signals in space or time. To help fill this knowledge gap, we used the toolkit of synthetic biology to systematically assay the effects of pairwise and triplet combinations of mutant genotypes on the expression pattern of a gene regulatory network expressed in Escherichia coli that interprets an inducer gradient across a spatial domain. We uncovered a preponderance of epistasis in both pairwise and triplet combinations that can switch in magnitude and sign across the inducer gradient to produce a greater diversity of expression pattern phenotypes than would be possible in the absence of such environment-dependent epistasis. We discuss our findings in the context of the evolution of hybrid incompatibilities and evolutionary novelties, arguing that environment-dependent epistasis is likely an important cause of both phenomena in gene regulatory networks.

evolutionary biology↗

bioRxiv2022

Synthetic genotype networks

Genotype networks are sets of genotypes connected by small mutational changes that share the same phenotype. They facilitate evolutionary innovation by enabling the exploration of different neighborhoods in genotype space. Genotype networks, first suggested by theoretical models, have been empirically confirmed for proteins and RNAs. Comparative studies also support their existence for gene regulatory networks (GRNs), but direct experimental evidence is lacking. Here, we report the construction of three interconnected genotype networks of synthetic GRNs producing three distinct gene expression phenotypes in Escherichia coli. These genotype networks, composed of over twenty different synthetic GRNs, provide robustness in face of mutations while enabling transitions to innovative phenotypes. Through realistic mathematical modeling, we quantify robustness and evolvability for the complete genotype-phenotype map and link these features mechanistically to GRN motifs. Our work thereby exemplifies how GRN evolution along genotype networks might be driving evolutionary innovation.

synthetic biology↗

bioRxiv2019

Controlling spatiotemporal pattern formation in a concentration gradient with a synthetic toggle switch

The formation of spatiotemporal patterns of gene expression is frequently guided by gradients of diffusible signaling molecules. The toggle switch subnetwork, composed of two cross-repressing transcription factors, is a common component of gene regulatory networks in charge of patterning, converting the continuous information provided by the gradient into discrete abutting stripes of gene expression. We present a synthetic biology framework to understand and characterize the spatiotemporal patterning properties of the toggle switch. To this end, we built a synthetic toggle switch controllable by diffusible molecules in Escherichia coli. We analyzed the patterning capabilities of the circuit by combining quantitative measurements with a mathematical reconstruction of the underlying dynamical system. The toggle switch can produce robust patterns with sharp boundaries, governed by bistability and hysteresis. We further demonstrate how the hysteresis, position, timing, and precision of the boundary can be controlled, highlighting the dynamical flexibility of the circuit.

synthetic biology↗