Biology subjects

Paulsson, J.

Publications and source records attributed to Paulsson, J..

12 recordsLinked to original sources

bioRxiv2026

Mapping Synthetic Circuit Design Space through Massively Parallel Measurement of Single-Cell Dynamics

Engineering predictable and robust gene-regulatory networks requires a quantitative understanding of how their dynamics depend on genetic design and environmental conditions. To investigate these relationships, we introduce BARRACUDA, a high-throughput platform that combines combinatorial assembly and programmable perturbations with lineage-resolved time-lapse imaging and optical pooled screening. Experiments using this platform follow approximately one million cell lineages, collectively sampling tens of thousands of circuit designs. We used BARRACUDA to investigate how the robustness of circuit behavior depends on molecular components and their interactions, combining quantitative dynamic measurements with precise genetic and environmental control. Antithetic integral feedback provided a model system linking a defined molecular architecture to theoretical predictions of robust adaptation. Through a hierarchy of increasingly complex libraries, we characterized individual components and used those measurements to model and interpret the dynamics of complete feedback circuits. Circuits built from wild-type inhibitory proteins rejected disturbances but also exhibited sustained overadaptation and fast disturbance-induced oscillations unexplained by a reduced control model. These discrepancies motivated a screen of de novo binders that retained inhibitory function while reducing host-stress response. Selected de novo binders supported strong feedback regulation and robust performance in the rebuilt circuits. Across more than 20,000 recovered feedback-circuit variants, binder identity and controller expression organized regimes of strong regulation and sustained oscillation. Counterintuitively, disturbances sometimes improved compensation rather than compromising it, consistent with nonlinear changes in feedback engagement. Together, these results show how structured libraries and massively parallel measurements of single-cell dynamics can test mechanistic theories across scales of circuit complexity, reveal missing biological interactions, and map the design space of gene-regulatory networks.

synthetic biology↗

bioRxiv2026

Migrations facilitated the cultural diffusion of megaliths in Neolithic Europe

Around 6700 BP (years before present), Neolithic Europe saw the emergence of megalithic monuments in northern France, followed by a rapid spread to the rest of the continent1. However, whether this was driven by the transmission of ideas (cultural diffusion) or the movement of people remains debated. To test whether this spread coincided with population movements, we reconstructed the population history of the European Neolithic ([~]6500-4800 BP) by analysing genomic data from 2963 ancient individuals, including 46 unpublished genomes from megalithic and related contexts. To assess the alternative scenario whereby megaliths spread through contact networks, as expected under cultural diffusion, we integrated archaeological evidence with radiocarbon chronologies to reconstruct past trading networks. We identify northern France, where megaliths first emerged, as a zone of genetic admixture and cultural influence from the two major early Neolithic divisions: the Cardial of the Western Mediterranean and the Linear Pottery traditions of central Europe. From this admixed population, we uncover several migrations associated with the formation of the Funnel Beaker Culture in north-central Europe and the neolithisation of Scandinavia and the British Isles. We find that megaliths were spread exclusively within regions impacted by Cardial-related genetic ancestry, but only in the British Isles and Ireland did their emergence coincide with the time of the migration. The temporal delay between the migrations and the spread of megaliths elsewhere reveals that neither population movements nor cultural diffusion alone fully explains the geospatial distribution of the megalithic tradition. Rather, megaliths diffused across different Neolithic groups whose long-distance trade networks formed in the wake of earlier population movements. Our findings highlight the need to move beyond simplistic interpretations that present migrations or cultural diffusion as isolated events bringing technological and cultural innovations.

evolutionary biology↗

bioRxiv2026

Division of labor during bacterial warfare

Division of labor is commonly associated with cooperative behavior, yet one of its most extreme forms occurs during bacterial warfare, where a subset of cells undergoes suicidal lysis to release toxins. Why bacteria divide labor among a few cells rather than producing toxin uniformly remains unknown. Here, we combine timelapse microscopy and simulations, to understand the division of labor during bacterial warfare using bacteriocin (colicin) production by the gut bacterium Escherichia coli as a model system. At the single-cell level, we find that lytic toxin production is a tightly regulated event: only cells that commit to lysis produce significant toxin and then lysis only occurs once a large amount of toxin has been made. This high threshold ensures each sacrifice delivers a large dose, which is released in a rapid burst from a lysing cell. While such burst-like release appears to provide no advantage over uniform labor in well-mixed conditions, we show it becomes extremely effective in spatially structured populations where the rapid release of toxin by one cell can generate lethal concentrations locally and eliminate competitors. Finally, we explain why the lysing fraction remains so small. While an increase in the producing cells increases toxin levels, it also increases the probability of local patch extinctions. The division of labor during bacterial warfare, therefore, enables powerful localized killing while safeguarding the population from self-destruction.

microbiology↗

bioRxiv2026

Inherited flagellar structures coordinate the onset of swimming among related E. coli cells

Bacteria commit to costly behaviors before they can benefit from them. In Escherichia coli, flagellar synthesis is expensive, yet the genes are expressed in stochastic pulses and only a small minority of pulses result in motility. Despite this noise, we find that lineage-related cells begin swimming at similar times, in groups of two to eight cells. Rare, high-amplitude pulses commit a single cell within one generation, but more commonly, pulses rise slowly and fail to trigger swimming before division. Rather than decaying, their output accumulates as cells inherit hook-basal bodies (HBBs) and continue to build new ones. This structural inheritance acts as a physical integrator, summing ongoing transcriptional activity across generations, so that an incomplete flagellar cascade is carried forward at division rather than lost. HBB accumulation sets when cells swim, and the anti-sigma factor FlgM sets how sharply. By holding late flagellar genes off until several HBBs have accumulated, FlgM makes motility depend steeply on early flagellar gene expression, so that relatives inheriting similar sets of HBBs become motile together. Without FlgM, this sharp dependence is largely lost, and activations of the full cascade do not reliably produce swimming. Together, our results show how the accumulation of a molecular machine integrates fluctuations, and how a checkpoint on its assembly sharpens them into a decision, setting both the speed and heritability of a behavioral trait.

systems biology↗

bioRxiv2025

Essentialome-Wide Multigenerational Imaging Reveals Mechanistic Origins of Cell Growth Laws

Escherichia coli is arguably the most thoroughly characterized organism, and yet [~]20% of its essential genes serve completely unknown functions,1,2 and many core quantitative physiological principles remain unexplained, including the famous nutrient growth law where cell volume seems to depend exponentially on growth rate. Here we develop a platform for massive, multi-generational Optical Pooled Screening (OPS)3-6, and apply it to link image-based phenotypes to genotypes for 133,000 CRISPRi knockdowns of essential genes, tracking tens of millions of lineages and analyzing 1.6 billion cells. Our multi-dimensional dynamic phenotypes correlate exceptionally well with known gene functions, allowing us to identify many unknown roles of essential genes. Quantifying the relation between growth and cell size in turn identifies three distinct variants of the bacterial growth laws, which we explain mechanistically by discovering a new role for (p)ppGpp and SpoT as a sensor of translation elongation. Finally, we propose and systematically test an exceedingly simple, passive mechanism for the nutrient growth law, based on triggering cell division through the accumulation of a protein that, unlike ribosomes, is not controlled by (p)ppGpp. The resulting hyperbolic growth law fits the data from our three variants even better than the previously proposed exponential relationship, and with fewer parameters.

systems biology↗

bioRxiv2025

Intracellular competition shapes plasmid population dynamics

Conflicts between levels of biological organization are central to evolution, from populations of multicellular organisms to selfish genetic elements in microbes. Plasmids are extrachromosomal, self-replicating genetic elements that underlie much of the evolutionary flexibility of bacteria. Evolving plasmids face selective pressures on their hosts, but also compete within the cell for replication, making them an ideal system for studying the joint dynamics of multilevel selection. While theory indicates that within-cell selection should matter for plasmid evolution, experimental measurement of within-cell plasmid fitness and its consequences has remained elusive. Here we measure the within-cell fitness of competing plasmids and characterize drift and selective dynamics. We achieve this by the controlled splitting of synthetic plasmid dimers to create balanced competition experiments. We find that incompatible plasmids co-occur for longer than expected due to methylation-based plasmid eclipsing. During this period of co-occurrence, less transcriptionally active plasmids display a within-cell selective advantage over their competing plasmids, leading to preferential fixation of silent plasmids. When the transcribed gene is beneficial to the cell, for example an antibiotic resistance gene, there is a cell-plasmid fitness tradeoff mediated by the dominance of the beneficial trait. Surprisingly, more dominant plasmid-encoded traits are less likely to fix but more likely to initially invade than less dominant traits. Taken together, our results show that plasmid evolution is driven by dynamics at two levels, with a transient, but critical, contribution of within-cell fitness.

evolutionary biology↗

bioRxiv2023

BatCRISPRi: Bacillus titratable CRISPRi for dynamic control in Bacillus subtilis

The discovery of new genes regulating essential biological processes has become increasingly important, and CRISPRi has emerged as a powerful tool for achieving this goal. This method has been used in many model organisms to decrease the expression of specific genes and assess their impact on phenotype. Pooled CRISPRi libraries in bacteria have been particularly useful in discovering new regulators of growth, division, and other biological processes. However, these libraries rely on the induction of dCas9 via an inducible promoter, which can be problematic due to promoter leakiness. This is a widespread phenomenon of any inducible promoter that can result in the unwanted downregulation of genes and the emergence of genetic suppressors when essential genes are knocked down. To overcome this issue, we have developed a novel strategy that eliminates dCas9 leakiness and enables reversible knockdown control using the rapamycin-dependent degron system in Bacillus subtilis. This degron system causes rapid degradation of dCas9, resulting in an almost instant reset of the system. Our results demonstrate that it is possible to achieve zero CRISPRi activity in the uninduced state and full activity in the induced state. This improved CRISPRi system will enable researchers to investigate phenotypic changes more effectively while reducing the undesirable effects of leaky expression and noise in their phenotypic data. Moreover, a rapid degradation system could serve as a tool for dynamic perturbation before compensation mechanisms or stress responses kick in. Finally, this approach can be adapted to other organisms and other promoter-inducible systems, potentially opening up strategies for tighter control of gene expression.

synthetic biology↗

bioRxiv2023

Cell wall fluidization by mechano-endopeptidases sets up a turgor-mediated volumetric pacemaker

The cell wall is an essential cellular component of bacteria and the target of many antibiotics. However, how bacteria regulate the rate of cell wall biosynthesis as growth rates change remains unresolved. In E. coli, cell wall growth was thought to proceed independently from turgor pressure1, the osmotic pressure that the cytoplasm exerts on the cell wall. Here, we uncover a striking increase of turgor pressure with growth rate. Modulating turgor pressure and measuring cell wall biosynthesis, we find that turgor pressure is directly controls the rate of cell wall biosynthesis. The picture that emerges is that turgor pressure is largely generated by counterions of negatively charged cellular biomass. The increase in turgor pressure with growth rates results from more ribosomes and therefore higher concentrations of negatively charged ribosomal RNA. Elegantly, the coupling between biomass composition, turgor pressure and cell wall biosynthesis simultaneously explains how bacteria achieve homeostasis of cytoplasmic crowding and how they regulate the rate of cell wall biosynthesis across growth rates.

microbiology↗

bioRxiv2020

Towards a translationally-independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nucleotide guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging ∼9 generations, a dCas12a oscillator design with 40-nucleotide CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ hours at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally-independent behavior, as the RNAs control each others’ transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Dynamic Regulation of Growth and Physiology of Microbes under Complex Changing Conditions

As bacteria transition from exponential to stationary phase they change greatly in size, morphology, growth and expression-profiles. These responses also vary between individual cells, but it has proven difficult to track cell lineages along the growth curve to determine the progression of events or correlations between how individual cells enter and exit dormancy. We developed a platform for tracking >105 parallel cell lineages in dense and changing cultures, independently validating that the imaged cells closely track batch populations. Initial applications show that for both Escherichia coli and Bacillus subtilis, growth changes from an adder mode in exponential phase to mixed adders-timers entering stationary phase, and then a near-perfect sizer upon exit - creating broadly distributed cell sizes in stationary phase but rapidly returning to narrowly distributed sizes upon exit. Furthermore, cells that undergo more divisions entering stationary phase suffer reduced survival after long periods of dormancy but are the only cells observed that persist against antibiotics.

microbiology↗

bioRxiv2018

Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator

The dynamics of the bacterial population that comprises the gut microbiota plays key roles in overall mammalian health. However, a detailed understanding of bacterial growth within the gut is limited by the inherent complexity and inaccessibility of the gut environment. Here, we deploy an improved synthetic genetic oscillator to investigate dynamics of bacterial colonization and growth in the mammalian gut under both healthy and disease conditions. The synthetic oscillator, when introduced into both Escherichia coli and Salmonella Typhimurium maintains regular oscillations with a constant period in generations across growth conditions. We determine the phase of oscillation from individual bacteria using image analysis of resultant colonies and thereby infer the number of cell divisions elapsed. In doing so, we demonstrate robust functionality and controllability of the oscillator circuits activity during bacterial growth in vitro, in a simulated murine gut microfluidic environment, and in vivo within the mouse gut. We determine different dynamics of bacterial colonization and growth in the gut under normal and inflammatory conditions. Our results show that a precise genetic oscillator can function in a complex environment and reveal single cell behavior under diverse conditions where disease may create otherwise impossible-to-quantify variability in growth across the population.

synthetic biology↗

bioRxiv2017

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is [≥]80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

systems biology↗