Richard M. Murray

Cell-free expression systems offer a method for the rapid prototyping of DNA circuits and functional protein synthesis. While crude extracts remain a black box with many components carrying out unknown reactions, PURE contains only the required transcription and translation components for protein production. All proteins and small molecules are at known concentrations, enabling detailed modeling of reliable computational predictions. However, there are few experimental data supporting the expression of target proteins for PURE-based models. In this work, we generalized the PURE detailed translation model for proteins with arbitrary amino acid compositions and lengths. We then built a chemical reaction network (CRN) for transcription in PURE, validating the transcription models using DNA expression for the malachite-green aptamer (MGapt) to measure RNA production. Lastly, we coupled the transcription and the generalized translation models to create a PURE protein synthesis model built purely of mass-action reactions. We used the combined model to capture the kinetics of MGapt and deGFP expressed from plasmids at various concentrations.

Cell-free expression systems offer a method for the rapid prototyping of DNA circuits and functional protein synthesis. While crude extracts remain a black box with many components carrying out unknown reactions, PURE contains only the required transcription and translation components for protein production. All proteins and small molecules are at known concentrations, enabling detailed modeling of reliable computational predictions. However, there are few experimental data supporting the expression of target proteins for PURE-based models. In this work, we generalized the PURE detailed translation model for proteins with arbitrary amino acid compositions and lengths. We then built a chemical reaction network (CRN) for transcription in PURE, validating the transcription models using DNA expression for the malachite-green aptamer (MGapt) to measure RNA production. Lastly, we coupled the transcription and the generalized translation models to create a PURE protein synthesis model built purely of mass-action reactions. We used the combined model to capture the kinetics of MGapt and deGFP expressed from plasmids at various concentrations.

Human posterior parietal cortex (PPC) is thought to play an important role in hand-eye coordination, yet the underlying encoding mechanisms remain uncertain. We recorded 412 single neurons across 11 sessions from motor cortex (MC; n=251) and PPC (n=161) in a single human participant performing a hand-eye (H-E) coordinated center-out task. While MC neurons showed little to no modulation by eye movements, 79% of PPC neurons had neural representations that were additively separable into independent hand- and eye-movement tuning curves. Due to this separability, neural representations could be separated and additively recomposed while maintaining structure similarity. Consequently, compositional decoders trained solely on single-effector movements could match the performance of decoders trained on coordinated H-E movements (hand: 66% vs 69%; eye: 34% vs 36%). These results show that, during simple center-out tasks, MC hand movement codes are unaffected by eye movements and that compositionality can be used to modularly decode H-E coordinated movements in PPC.

Steinernema species are soil-dwelling, insect-parasitic nematodes that maintain species-specific associations with Xenorhabdus symbiotic bacteria, which are packaged within anterior intestinal pockets during the infective juvenile (IJ) stage. While these nematodes can persist in soil for months while seeking insect hosts, their interactions with environmental microbes beyond their native symbionts remain poorly understood. Here, we describe a previously uncharacterized interaction between Escherichia coli Nissle 1917 (EcN) and Steinernema hermaphroditum. EcN cells are enclosed and lysed within multiple pairs of putative coelomocytes, suggesting microbial endocytosis by host cells. During the IJ stage, EcN localizes to posterior intestinal compartments and the inter-cuticular space, where cells proliferate, aggregate and subsequently lyse. Bacterially expressed proteins persist within the nematode cuticle for over 8 weeks in non-sterile soil. These findings reveal sequential stages of environmental bacterial colonization associated with host immune responses distinct from mutualistic symbiosis. This work establishes a model for understanding nematode and environmental microbe interactions and highlights opportunities to deliver bacterially expressed molecules for environmental biosensing and biocontrol applications.

Per-and polyfluoroalkyl substances (PFAS) are persistent, bioaccumulative environmental contaminants that pose significant risks to human health and water quality. Among them, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are highly regulated due to their toxicity and widespread occurrence at trace levels in drinking water. The need for rapid, sensitive, and field-deployable detection technologies has motivated the development of alternative sensing strategies beyond conventional laboratory-based methods. A porous metalorganic framework (MOF) incorporating gold nanoparticles is integrated onto microfabricated electrodes, enabling dual-mode detection via electrochemical impedance spectroscopy and voltammetry. It offers high surface area, tunable pore structures, and chemically adaptable metal nodes, enabling selective interactions with PFOA through electrostatic and hydrophobic mechanisms. Structural and electrochemical characterization confirmed the integrity and uniformity of the Au-Cr-MIL-101 nanocomposite and elucidated its molecular interaction with per-and polyfluoroalkyl substances (PFAS). The modified electrodes exhibit strong affinity toward long-chain PFAS, producing concentration-dependent changes in charge-transfer resistance and current response. Validation in river water samples confirms robust performance in complex matrices. This work highlights the potential of MOF-based electrochemical sensors as portable tools for rapid PFAS screening and real-time water quality monitoring.

Abstract Cell-free expression systems offer a method for rapid prototyping of DNA circuits and functional protein synthesis. While crude extracts remain a black box with many components carrying out unknown reactions, PURE contains only the required transcription and translation components for protein production. All proteins and small molecules are at known concentrations, enabling detailed modeling for reliable computational predictions. However, there is little to no experimental data supporting the expression of target proteins for PURE-based models. In this work, we generalized the PURE detailed translation model for proteins with arbitrary amino acid compositions and lengths. We then built a chemical reaction network for transcription in PURE, validating the transcription models using DNA expression for the malachite-green aptamer (MGapt) to measure mRNA production. Lastly, we coupled the transcription and the generalized translation models to create a PURE protein synthesis model built purely of mass-action reactions. We used the combined model to capture the kinetics of MGapt and deGFP expressed from plasmids at varying concentrations.

Immune cells naturally secrete extracellular antigen-presenting vesicles (APVs) displaying peptide:MHC complexes to facilitate the initiation, expansion, maintenance, or silencing of immune responses. Previous work has sought to manufacture and purify these vesicles for cell-free immunotherapies. In this study, APV assembly and release is achieved in nonimmune cells by transfecting a single-chain heterotrimer (SCT) peptide major histocompatibility complex I (pMHCI) construct containing an ESCRT- and ALIX-binding region (EABR) sequence appended to the cytoplasmic tail; this EABR sequence recruits ESCRT proteins to induce the budding of APVs displaying SCT pMHCI. A comparison of multiple pMHCI constructs shows that inducing the release of APVs by the addition of an EABR sequence generalizes across SCT pMHCI constructs. Purified pMHCI/EABR APVs selectively stimulate IFN-γ release from T cells presenting their cognate T cell receptor, demonstrating the potential use of these vesicles as a form of cell-free immunotherapy.

Luminol-based Electrochemiluminescence (ECL) generates weak signals in neutral media and typically requires H 2 O 2 as a coreactant. However, H 2 O 2 instability and the need for on-site addition hinder real-time diagnostic applications. Compact integrated sensing platforms are ideal for point-of-care (POC) testing due to portability, low sample requirements, and multiplexing. However, as sensor dimensions decrease, the low light emission issue in ECL becomes severe. We introduce a novel fully integrated miniaturized silicon device consisting of three sensors comprised of unique entwined micro-spiral electrodes in a generator-collector configuration. This enables highly sensitive, coreactant-free multiplexed sensing at physiological pH by accelerating in-situ reactive oxygen species production and boosting ECL intensity. Systematic optimization of electrode geometry (gaps and widths) yields an 11-fold improvement in ECL signal compared to a single-electrode setup, as well as excellent reproducibility and stability. In addition to Trolox and H 2 O 2 detection, the platform demonstrates multiplex immunosensing through selective functionalization of the collector electrodes with chitosan nanocomposites, followed by Protein A/G-assisted immobilization of anti-IgG antibodies with peptide-based antifouling. The immunosensors exhibit high analytical performance (linear range: 0.001-100 pg·mL -1 , detection limit: 0.8 fg·mL -1 ) with excellent reproducibility and reliability in 25% fetal bovine serum, highlighting the platform’s potential for sensitive, coreactant-free multi-analyte POC diagnostics.

Abstract Luminol-based Electrochemiluminescence (ECL) generates weak signals in neutral media and typically requires H 2 O 2 as a coreactant. However, H 2 O 2 instability and the need for on-site addition hinder real-time diagnostic applications. Compact integrated sensing platforms are ideal for point-of-care (POC) testing due to portability, low sample requirements, and multiplexing. However, as sensor dimensions decrease, the low light emission issue in ECL becomes severe. We introduce a novel fully integrated miniaturized silicon device consisting of three sensors comprised of unique entwined micro-spiral electrodes in a generator-collector configuration. This enables highly sensitive, coreactant-free multiplexed sensing at physiological pH by accelerating in-situ reactive oxygen species production and boosting ECL intensity. Systematic optimization of electrode geometry (gaps and widths) yields an 11-fold improvement in ECL signal compared to a single-electrode setup, as well as excellent reproducibility and stability. In addition to Trolox and H 2 O 2 detection, the platform demonstrates multiplex immunosensing through selective functionalization of the collector electrodes with chitosan nanocomposites, followed by Protein A/G-assisted immobilization of anti-IgG antibodies with peptide-based antifouling. The immunosensors exhibit high analytical performance (linear range: 0.001-100 pg·mL −1 , detection limit: 0.8 fg·mL −1 ) with excellent reproducibility and reliability in 25% fetal bovine serum, highlighting the platform’s potential for sensitive, coreactant-free multi-analyte POC diagnostics.

Quadrotor Morpho-Transition, or the act of transitioning from air to ground through mid-air transformation, involves complex aerodynamic interactions and a need to operate near actuator saturation, complicating controller design. In recent work, morpho-transition has been studied from a model-based control perspective, but these approaches remain limited due to unmodeled dynamics and the requirement for planning through contacts. Here, we train an end-to-end Reinforcement Learning (RL) controller to learn a morpho-transition policy and demonstrate successful transfer to hardware. We find that the RL control policy achieves agile landing, but only transfers to hardware if motor dynamics and observation delays are taken into account. On the other hand, a baseline MPC controller transfers out-of-the-box without knowledge of the actuator dynamics and delays, at the cost of reduced recovery from disturbances in the event of unknown actuator failures. Our work opens the way for more robust control of agile in-flight quadrotor maneuvers that require mid-air transformation. Video; Code.

In safety-critical robotic systems, perception is tasked with representing the environment to effectively guide decision-making and plays a crucial role in ensuring that the overall system meets its requirements. To quantitatively assess the impact of object detection and classification errors on system-level performance, we present a rigorous formalism for a model of detection error, and probabilistically reason about the satisfaction of regular-safety temporal logic requirements at the system level. We also show how standard evaluation metrics for object detection, such as confusion matrices, can be represented as models of detection error, which enables the computation of probabilistic satisfaction of system-level specifications. However, traditional confusion matrices treat all detections equally, without considering their relevance to the system-level task. To address this limitation, we propose novel evaluation metrics for object detection that are informed by both the system-level task and the downstream control logic, enabling a more context-appropriate evaluation of detection models. We identify logic-based formulas relevant to the downstream control and system-level specifications and use these formulas to define a logic-based evaluation metric for object detection and classification. These logic-based metrics result in less conservative assessments of system-level performance. Finally, we demonstrate our approach on a car-pedestrian example with a leaderboard PointPillars model evaluated on the nuScenes dataset, and validate probabilistic system-level guarantees in simulation.

Xenorhabdus griffiniae is a bacterium that lives inside the intestine of the entomopathogenic nematode Steinernema hermaphroditum and partners with the nematode to infect and kill insect larvae in soil. The construction of gene circuits, such as reporters, in X. griffiniae would provide tools to study and better understand the symbiotic relationship it has with its host. However, because X. griffiniae is not a model organism, information about gene circuit construction in X. griffiniae is limited. We developed and characterized a DNA part library similar to the CIDAR MoClo extension library for E. coli to allow more efficient construction of genetic circuits in X. griffiniae . TurboRFP expressing strains with different constitutive Anderson promoters and different ribosome binding sites (RBS) were constructed to quantify promoter and RBS strengths in X. griffiniae . Furthermore, two fluorescent proteins sfGFP and sfYFP as well as the bioluminescent luxCDABE operon were added to the part library and successfully expressed in X. griffiniae . We then used the characterized parts of the cell to build and characterize IPTG inducible constructs.

Abstract Some biochemical functions can be performed more efficiently when split into different tasks, each performed by distinct strains within a microbial consortium. Due to the distinct growth dynamics of each strain, uncontrolled consortia have unstable composition dynamics, leading to the rapid loss of the community level function. Several approaches have been developed to stabilize consortia, with most relying on communicated-mediated growth and death feedback. These approaches require accurate communication between strains to maintain control, something which is not guaranteed under non-well-mixed conditions. As such, these methods are of limited utility in consortia applications with poorly mixed environments e.g. industrial scale bioreactors or soil. Here, inspired by microbial phase variation dynamics, we introduce an alternative, communication-free approach in which a set of genetically identical cells switch stochastically between distinct phenotypes. In this scheme, the population composition is dynamically stable and determined by the rates of transitions between states. Mathematical modeling indicates that this approach can stabilize consortia. Experimentally, we used reversible DNA inversions catalyzed by serine recombinases to implement a dynamic consortium. We then characterized the dynamic properties of the consortium at the single cell and bulk levels, and demonstrated control in 2- and 3-state consortia. These results provide a composition control approach that does not rely on cell to cell communication, providing a foundation for deployment of engineered consortia in complex, and sometimes non-mixed, environments such as industrial scale bioreactors or the human gut.

Identifying the cause of a system-level failure in a cyber-physical system (CPS) can be like tracing a needle in a haystack. This paper approaches the problem by assuming that the CPS has been designed compositionally and that each component in the system is associated with an assume-guarantee contract. We exploit recent advances in contract-based design that show how to compute the contract for the entire system using the component-level contracts. When presented with a system-level failure, our approach is able to efficiently identify the components that are responsible for the system-level failure together with the specific predicates in those components' specifications that are involved in the fault. We implemented this approach using Pacti and demonstrate it through illustrative examples inspired by an autonomous vehicle in the DARPA urban challenge.

Abstract Steinernema species are soil-dwelling and insect-parasitic nematodes that associate with symbiotic Xenorhabdus bacteria. During the infective juvenile (IJ) stage , Steinernema nematodes package species-specific Xenorhabdus bacteria in the anterior intestinal pockets. The nematodes can survive in the soil for months as they seek insect prey. The mechanisms of how these nematodes associate with environmental microbes other than their Xenorhabdus symbionts is barely known. Here, we report a new mechanism of E. coli Nissle (EcN) association with the nematode Steinernema hermaphroditum. We show that EcN cells are enclosed and lysed in at least four pairs of coelomocytes, suggesting these immune cells respond to bacterial invasion. During the IJ stage of nematode development, EcN cells localize to posterior intestinal vacuoles and enter the inter-cuticular space, where they proliferate, aggregate, then lyse. EcN cells expressed proteins in the cell lysates were maintained in the nematode cuticle over eight weeks in non-sterile soil. These observations suggest sequential steps of EcN colonization in the host nematodes involving an immune response that is distinctive from interactions with mutualistic symbiont. Our work establishes a novel framework of nematode-bacteria interaction with potential applications in environmental bioengineering.

Cell-free expression systems have gained considerable attention for their potential in biomanufacturing, biosensing, and circuit prototyping. All these endeavors make use of the innate metabolic activity of cell lysates. However, our knowledge of the underlying nature of cell-free metabolism remains lacking. In this work, we use non-targeted mass spectrometry to generate time-course measurements of small molecules in E. coli cell lysates (metabolomics) during cell-free protein synthesis (CFPS) to show that the majority of E. coli 's metabolism is active in cell lysate. Furthermore, these data indicate that protein synthesis is a relatively small metabolic burden on cell lysates compared to the background metabolism, and that the build-up of multiple metabolic waste products is fundamentally responsible for stopping cell-free protein expression as opposed to depletion of fuel sources. We use these insights coupled with high-throughput CFPS experiments to develop a foundational coarse-grained mechanistic model of CFPS which we show can be easily recalibrated using Bayesian parameter inference techniques to provide accurate models across hundreds of novel experimental conditions. This work provides new experimental insights into the effects of cell-free metabolism on CFPS and establishes a novel framework for leveraging existing CFPS models in new experimental contexts, opening the avenue to future cell-free applications aimed at building complex systems, from multilayered biological circuits to synthetic biological cells.

Quadrotor Morpho-Transition, or the act of transitioning from air to ground through mid-air transformation, involves complex aerodynamic interactions and a need to operate near actuator saturation, complicating controller design. In recent work, morpho-transition has been studied from a model-based control perspective, but these approaches remain limited due to unmodeled dynamics and the requirement for planning through contacts. Here, we train an end-to-end Reinforcement Learning (RL) controller to learn a morpho-transition policy and demonstrate successful transfer to hardware. We find that the RL control policy achieves agile landing, but only transfers to hardware if motor dynamics and observation delays are taken into account. On the other hand, a baseline MPC controller transfers out-of-the-box without knowledge of the actuator dynamics and delays, at the cost of reduced recovery from disturbances in the event of unknown actuator failures. Our work opens the way for more robust control of agile in-flight quadrotor maneuvers that require mid-air transformation.

Abstract Xenorhabdus griffiniae is a bacterium that lives inside the intestine of the entomopathogenic nematode Steinernema hermaphroditum and partners with the nematode to infect and kill insect larvae in soil. The construction of gene circuits, like reporters, in X. griffiniae would provide tools to study and better understand the symbiotic relationship it has with its host. However, because X. griffiniae is not a model organism, information about gene circuit construction in X. griffiniae is limited. We develop and characterize a DNA part library similar to the CIDAR MoClo extension library for E. coli to allow more efficient construction of genetic circuits in X. griffiniae . TurboRFP expressing strains with different constitutive Anderson promoters and different ribosome binding sites (RBS) were constructed to quantify promoter and RBS strengths in X. griffiniae . Furthermore, two fluorescent proteins sfGFP and sfYFP, as well as the bioluminescent luxCDABE operon were added to the part library and successfully expressed in X. griffiniae . We then used the characterized parts to build and characterize IPTG inducible constructs.

Designing ground-aerial robots is challenging due to the increased actuation requirements which can lead to added weight and reduced locomotion efficiency. Morphobots mitigate this by combining actuators into multi-functional groups and leveraging ground transformation to achieve different locomotion modes. However, transforming on the ground requires dealing with the complexity of ground-vehicle interactions during morphing, limiting applicability on rough terrain. Mid-air transformation offers a solution to this issue but demands operating near or beyond actuator limits while managing complex aerodynamic forces. We address this problem by introducing the Aerially Transforming Morphobot (ATMO), a robot which transforms near the ground achieving smooth transition between aerial and ground modes. To achieve this, we leverage the near ground aerodynamics, uncovered by experimental load cell testing, and stabilize the system using a model-predictive controller that adapts to ground proximity and body shape. The system is validated through numerous experimental demonstrations. We find that ATMO can land smoothly at body postures past its actuator saturation limits by virtue of the uncovered ground-effect. Ioannis Mandralis and colleagues present the Aerially Transforming Morphobot (ATMO), a robot that morphs mid-air to transition between flight and ground locomotion. By harnessing near-ground aerodynamics, ATMO achieves smooth landings beyond actuator limits, unlocking new possibilities for hybrid ground-aerial mobility.

High Resolution Image Download MS PowerPoint Slide Bacteria innately monitor their environment by dynamically regulating gene expression to respond to fluctuating conditions. Through synthetic biology, we can harness this natural capability to design cell-based sensors. Bacillus megaterium, a soil bacterium, stands out due to its remarkable heavy metal tolerance and sporulation ability, making it an ideal candidate for heavy metal detection with low transportation costs. However, challenges persist: the synthetic biology toolkit for this strain is underdeveloped, and conventional whole-cell sensors necessitate specialized laboratory equipment to read the output. In our study, we have genetically modified B. megaterium for arsenic detection and established a detection threshold below the EPA’s recommendation of 10 ppb for drinking water in both vegetative and spore forms. Additionally, we have integrated both engineered B. megaterium living cells and spores with a complementary metal-oxide-semiconductor (CMOS) chip, providing a proof-of-concept for field-deployable arsenic detection. We show that the limit of detection (LOD) of our integrated sensor is within the range to test arsenic levels in soil and food. As a proof of concept, this work paves the way for the deployment of our sensor in resource-limited settings, ensuring real-time arsenic detection in challenging environments.

The One-Pot PURE ( P rotein synthesis U sing R ecombinant E lements) system simplifies the preparation of traditional PURE systems by coculturing and purifying 36 essential proteins for gene expression in a single step, enhancing accessibility and affordability for widespread laboratory adoption and customization. However, replicating this protocol to match the productivity of traditional PURE systems can take considerable time and effort due to uncharacterized variability. In this work, we observed unstable PURE protein expression in the original One-Pot PURE strains, E. coli M15/pREP4 and BL21(DE3), and addressed this issue using glucose-mediated catabolite repression to minimize burdensome background expression. We also identified several limitations making the M15/pREP4 strain unsuitable for PURE protein expression, including coculture incompatibility with BL21(DE3) and uncharacterized proteolytic activity. We showed that consolidating all expression vectors into a protease-deficient BL21(DE3) strain minimized proteolysis, led to more uniform coculture cell growth at the time of induction, and improved the stoichiometry of critical translation initiation factors in the final PURE mixture for efficient cell-free protein production. In addition to optimizing the One-Pot PURE protein composition, we found that variations in commercial energy solution formulations could compensate for suboptimal PURE protein stoichiometry. Notably, altering the source of E. coli tRNAs in the energy solution alone led to significant differences in the expression capacity of cell-free reactions, highlighting the importance of tRNA codon usage in influencing protein expression yield. Taken together, this work systematically investigates the proteome and biochemical factors influencing the One-Pot PURE system productivity, offering insights to enhance its robustness and adaptability across laboratories.

Designing ground-aerial robots is challenging due to the increased actuation requirements which can lead to added weight and reduced locomotion efficiency. Morphobots mitigate this by combining actuators into multi-functional groups and leveraging ground transformation to achieve different locomotion modes. However, transforming on the ground requires dealing with the complexity of ground-vehicle interactions during morphing, limiting applicability on rough terrain. Mid-air transformation offers a solution to this issue but demands operating near or beyond actuator limits while managing complex aerodynamic forces. We address this problem by introducing the Aerially Transforming Morphobot (ATMO), a robot which transforms near the ground achieving smooth transition between aerial and ground modes. To achieve this, we leverage the near ground aerodynamics, uncovered by experimental load cell testing, and stabilize the system using a model-predictive controller that adapts to ground proximity and body shape. The system is validated through numerous experimental demonstrations. We find that ATMO can land smoothly at body postures past its actuator saturation limits by virtue of the uncovered ground-effect.

Duplication cysts are rare congenital cystic lesions that form along the gastrointestinal tract at various locations. They may be asymptomatic, present with ambiguous symptoms, or mimic other clinical presentations, such as acute appendicitis. We present a unique case of an 11- year-old boy with a preliminary diagnosis of acute perforated appendicitis and abdominal abscess; however, the patient’s appendix was unremarkable during surgery, and he was later found to have an intraluminal duplication cyst within the distal ilium extending into the ileocecal valve. Follow-up imaging with abdominal ultrasound helped guide the diagnosis, and an ileocecal resection proved to be curative. Keywords: Enteric duplication cyst, intraluminal duplication cyst, appendicitis, ultrasound, case report

Abstract Immune cells naturally secrete extracellular antigen-presenting vesicles (APVs) displaying peptide:MHC complexes to facilitate the initiation, expansion, maintenance, or silencing of immune responses. Previous work has sought to manufacture and purify these vesicles for cell-free immunotherapies. In this study, APV assembly and release is achieved in non-immune cells by transfecting HEK293T or Expi293F cells with a single-chain heterotrimer (SCT) peptide/major histocompatibility complex I (pMHCI) construct containing an ESCRT- and ALIX-binding region (EABR) sequence appended to the cytoplasmic tail; this EABR sequence recruits ESCRT proteins to induce the budding of APVs displaying SCT pMHCI. A comparison of multiple pMHCI constructs shows that inducing the release of APVs by the addition of an EABR sequence generalizes across SCT pMHCI constructs. Purified pMHCI/EABR APVs selectively stimulate IFN-γ release from T cells presenting their cognate T cell receptor, demonstrating the potential use of these vesicles as a form of cell-free immunotherapy. Significance Statement Immune cells are known to naturally release pMHC-displaying extracellular vesicles (EVs), called antigen-presenting vesicles (APVs), which can orchestrate immune responses either directly or with the aid of antigen-presenting cells (APCs). For decades, researchers have pursued ways to replicate these APVs for immunotherapy by using chemically modified nanoparticles or by engineering the increased expression of APVs from immune cells which are typically low yield. Here we presents a broadly applicable platform for generating high concentrations of pMHCI-displaying APVs that can selectively modulate T cells, demonstrating a significant advance in the engineering of APVs for cell-free immunotherapy. The APVs presented here, and related APVs, could be translated into clinical therapies for modulating cancer progression or regulating autoimmunity in addition to their use as a tool to help characterize how endogenous extracellular vesicles influence the immune system.

Designing tests for autonomous systems is challenging due to their complexity. This work proposes a flow-based approach for reactive test synthesis from temporal logic specifications, enabling the synthesis of test environments consisting of static and reactive obstacles, and dynamic test agents that can transition between states. These specifications describe desired test behavior, including system requirements as well as a test objective not revealed to the system. The synthesized test strategy places restrictions on system actions in closed-loop with system behavior, accomplishing the test objective while ensuring realizability of the system's objective without aiding it (a general-sum setting). Automata theory and flow networks are leveraged to formulate a mixed-integer linear program (MILP) for test synthesis. For a dynamic test agent, the agent strategy is synthesized for a generalized reactivity of rank 1 (GR(1)) specification constructed from the MILP solution. If this solution is not realizable with the test agent's dynamics, we add a counterexample-guided constraint to re-solve the MILP until a strategy is found. This flow-based, reactive test synthesis is conducted offline and is agnostic to the system controller. Finally, the resulting test strategy is demonstrated in simulation and hardware experiments on a pair of quadrupedal robots for a variety of specifications.

Bacteria genetically engineered to execute defined therapeutic and diagnostic functions in physiological settings can be applied to colonize the human microbiome, providing in situ surveillance and conditional disease modulation. However, many engineered microbes can only respond to single-input environmental factors, limiting their tunability, precision, and effectiveness as living diagnostic and therapeutic systems. For engineering microbes to improve complex chronic disorders such as inflammatory bowel disease, the bacteria must respond to combinations of stimuli in the proper context and time. This work implements a previously characterized split activator AND logic gate in the probiotic Escherichia coli strain Nissle 1917 (EcN). Our system can respond to two input signals: the inflammatory biomarker tetrathionate and a second input signal, anhydrotetracycline (aTc), for manual control. We report 4–6 fold induction with a minimal leak when the two chemical signals are present. We model the AND gate dynamics using chemical reaction networks and tune parameters in silico to identify critical perturbations that affect our circuit’s selectivity. Finally, we engineer the optimized AND gate to secrete a therapeutic anti-inflammatory cytokine IL-22 using the hemolysin secretion pathway in the probiotic E. coli strain. We used a germ-free transwell model of the human gut epithelium to show that our engineering bacteria produce similar host cytokine responses compared to recombinant cytokine. Our study presents a scalable workflow to engineer cytokine-secreting microbes driven by logical signal processing. It demonstrates the feasibility of IL-22 derived from probiotic EcN with minimal off-target effects in a gut epithelial context.

We propose an adversarial, time-varying test-synthesis procedure for safety-critical systems <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">without requiring specific knowledge of the underlying controller steering the system</i>. Specifically, our approach codifies the system objective as a timed reach-avoid specification. Then, by coupling control barrier functions with this class of specifications, we construct an instantaneous difficulty metric whose minimizer corresponds to the most difficult test at that system state. By defining tests as the minimizer of this difficulty metric over the space of allowable tests, we provably identify realizable and maximally difficult tests of system behavior. Finally, we develop this test-synthesis procedure for both continuous and discrete-time systems and showcase our test-synthesis procedure on simulated and hardware examples.

Cell-free transcription–translation (TX–TL) systems have been used for diverse applications, but their performance and scope are limited by variability and poor predictability. To understand the drivers of this variability, we explored the effects of metabolic perturbations to an Escherichia coli ( E. coli ) Rosetta2 TX–TL system. We targeted three classes of molecules: energy molecules, in the form of nucleotide triphosphates (NTPs); central carbon “fuel” molecules, which regenerate NTPs; and magnesium ions (Mg 2+ ). Using malachite green mRNA aptamer (MG aptamer) and destabilized enhanced green fluorescent protein (deGFP) as transcriptional and translational readouts, respectively, we report the presence of a trade-off between optimizing total protein yield and optimizing total mRNA yield, as measured by integrating the area under the curve for mRNA time-course dynamics. We found that a system’s position along the trade-off curve is strongly determined by Mg 2+ concentration, fuel type and concentration, and cell lysate preparation and that variability can be reduced by modulating these components. Our results further suggest that the trade-off arises from limitations in translation regulation and inefficient energy regeneration. This work advances our understanding of the effects of fuel and energy metabolism on TX–TL in cell-free systems and lays a foundation for improving TX–TL performance, lifetime, standardization, and prediction.

Contract-based design is a method to facilitate modular design of systems. While there has been substantial progress on the theory of contracts, there has been less progress on practical algorithms for the algebraic operations in the theory. In this article, we present (1) principles to implement a contract-based design tool at scale and (2) Pacti, a tool that can efficiently compute these operations. We illustrate the use of Pacti in a variety of case studies.

and in lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect RNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system's chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.

Median arcuate ligament syndrome (MALS) is a rare condition in which the median arcuate ligament (MAL) exerts external compression on the celiac trunk. Most cases are asymptomatic and diagnosed incidentally on radiographic imaging; however, some patients may experience gastrointestinal (GI) symptoms related to foregut ischemia and/or celiac neuropathy. In the following case, we present a patient with hemorrhagic peptic ulcer disease of the duodenum, which resulted in episodes of hemodynamic instability requiring multiple blood transfusions. Upon attempted transarterial angioembolization of the gastroduodenal artery (GDA), celiac stenosis and retrograde arterial flow from the superior mesenteric artery confirmed the presence of MALS. This rendered GDA angioembolization a contraindication, as the GDA became the dominant arterial supply for the distal celiac organs. The patient then received open surgical MAL release with concurrent surgical ligation of the hemorrhaging duodenal artery, which resolved his symptoms without the need for further intervention.

Abstract Bacteria innately monitor their environment by dynamically regulating gene expression to respond to fluctuating conditions. Through synthetic biology, we can harness this natural capability to design cell-based sensors. Bacillus megaterium , a soil bacterium, stands out due to its remarkable heavy metal tolerance and sporulation ability, making it an ideal candidate for heavy metal detection with low transportation costs. However, challenges persist: the synthetic biology toolkit for this strain is underdeveloped and conventional whole-cell sensors necessitate specialized laboratory equipment to read the output. In our study, we genetically modified B. megaterium for arsenic detection, establishing a detection threshold below the EPA recommendation of 10 ppb for drinking water in both vegetative cell form and spore form. Additionally, we integrated both engineered B. megaterium living cells and spores with CMOS chip for field-deployable arsenic detection. We show that the limit of detection of our integrated sensor is applicable in soil and air arsenic contamination testing. As a proof of concept, this work paves the way for deploying our sensor in resource-limited settings, ensuring real-time arsenic detection in challenging environments. Abstract Figure

We consider the problem of estimating the subset of test conditions under which a simplified model—or set of simplified models—accurately approximates the behavior of a true system. We approach the problem by proposing a compact set of possible test conditions, and an unknown but samplable continous validity function over that set that quantifies the accuracy of the model under each possible condition. We propose a novel Bayes estimator that optimally directs function sampling to greedily minimize the expected posterior misclassification rate of the valid set, which we call minimum posterior misclassification sampling (GP-MPM), and we show that the the method can be extended to approximate the valid sets of a partially ordered set of models, with sample complexity growing sublinearly with the number of models. In testing against a safety-focused model, we show that the algorithm's estimated valid set approaches the true valid set much more quickly than undirected sampling, even with small sample sizes.

ABSTRACT The One-Pot PURE ( P rotein synthesis U sing R ecombinant E lements) system simplifies the preparation of traditional PURE systems by co-culturing and purifying 36 essential proteins for gene expression in a single step, thereby improving accessibility and affordability for widespread laboratory adoption and customization. However, replicating this protocol to match the productivity of traditional PURE systems can take considerable time and effort due to uncharacterized variability in the system’s biochemical composition. In this work, we observed unstable PURE protein expression in E. coli strains M15/pREP4 and BL21(DE3) and addressed this using glucose-mediated catabolite repression to minimize burdensome background expression. We also identified differences in optimal protein induction timing between these two strains, leading to growth incompatibility in co-culture, and observed proteolysis of PURE proteins expressed in M15/pREP4. We showed that consolidating all expression vectors into a protease-deficient BL21(DE3) strain could minimize proteolysis. This single-strain system also led to more uniform cell growth at the time of protein induction, improving the stoichiometry of critical translation initiation factors in the PURE reaction for efficient protein production. In addition to optimizing One-Pot PURE protein composition, we found that variations in commercial energy solution formulations could compensate for suboptimal PURE protein stoichiometry. Moreover, our study revealed significant differences in the expression capacity of commercially available E. coli tRNAs, suggesting the potential of optimizing tRNA codons to improve protein translation. Taken together, this work highlights the complex biochemical interplay influencing protein expression capacity in the One-Pot PURE system and presents strategies to improve its robustness and productivity.

Designing tests to evaluate if a given autonomous system satisfies complex specifications is challenging due to the complexity of these systems. This work proposes a flow-based approach for reactive test synthesis from temporal logic specifications, enabling the synthesis of test environments consisting of static and reactive obstacles and dynamic test agents. The temporal logic specifications describe desired test behavior, including system requirements as well as a test objective that is not revealed to the system. The synthesized test strategy places restrictions on system actions in reaction to the system state. The tests are minimally restrictive and accomplish the test objective while ensuring realizability of the system's objective without aiding it (semi-cooperative setting). Automata theory and flow networks are leveraged to formulate a mixed-integer linear program (MILP) to synthesize the test strategy. For a dynamic test agent, the agent strategy is synthesized for a GR(1) specification constructed from the solution of the MILP. If the specification is unrealizable by the dynamics of the test agent, a counterexample-guided approach is used to resolve the MILP until a strategy is found. This flow-based, reactive test synthesis is conducted offline and is agnostic to the system controller. Finally, the resulting test strategy is demonstrated in simulation and experimentally on a pair of quadrupedal robots for a variety of specifications.

Abstract The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect mRNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system’s chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.

In vitro transcription–translation (TX–TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX–TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens . All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX–TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX–TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX–TL-derived promoter strength and the in vivo promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design–build–test cycle.

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty in ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2–79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25–100 μM without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8–8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporter’s performance in shaken liquid culture predicts its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally aware design process provides an example of how laboratory bioengineering efforts can generate microbes with a greater promise to function reliably in their applied contexts.