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Synthetic circuit for exact adaptation and fold-change detection + (Biological organisms use their sensory sys … Biological organisms use their sensory systems to detect changes in their environment. The ability of sensory systems to adapt to static inputs allows wide dynamic range as well as sensitivity to input changes including fold-change detection, a response that de- pends only on fold changes in input, and not on ab- solute changes. This input scale invariance underlies an important strategy for search that depends solely on the spatial profile of the input. Synthetic efforts to reproduce the architecture and response of cellu- lar circuits provide an important step to foster under- standing at the molecular level. We report the bottom- up assembly of biochemical systems that show exact adaptation and fold-change detection. Using a malachite green aptamer as the output, a synthetic tran- scriptional circuit with the connectivity of an incoherent feed-forward loop motif exhibits pulse generation and exact adaptation. A simple mathematical model was used to assess the amplitude and duration of pulse response as well as the parameter regimes required for fold-change detection. Upon parameter tuning, this synthetic circuit exhibits fold-change detection for four successive rounds of two-fold input changes. The experimental realization of fold-change detection circuit highlights the programmability of transcriptional switches and the ability to obtain predictive dynamical systems in a cell-free environment for technological applications.nvironment for technological applications.)
Systematic Design and Implementation of a Novel Synthetic Fold-Change Detector Biocircuit In Vivo + (Biological signaling systems not only dete … Biological signaling systems not only detect the absolute levels of the signals, but are also able to sense the fold-changes of the signals. The ability to detect fold-changes provides a powerful tool for biological organisms to adapt to the changes in environment. Here we present the first novel syn- thetic fold-change detector (FCD) circuit built from ground up in vivo. We systematically designed the FCD circuit in silico, prototyped it in cell-free transcription-translation platform (TX-TL), and eventually implemented it in E. coli cells. We were able to show that the FCD circuit can not only generate pulse-like behavior in response to input, but also produce the same pulse response with inputs of the same fold-change, despite of di�erent absolute signal levels.espite of di�erent absolute signal levels.)
An analytical approach to bistable biological circuit discrimination using real algebraic geometry + (Biomolecular circuits with two distinct an … Biomolecular circuits with two distinct and stable steady states have been identified as essential components in a wide range of biological networks, with a variety of mechanisms and topologies giving rise to their important bistable property. Understanding the differences between circuit implementations is an important question, particularly for the synthetic biologist faced with determining which bistable circuit design out of many is best for their specific application. In this work we explore the applicability of Sturm's theorem—a tool from nineteenth-century real algebraic geometry—to comparing ‘functionally equivalent’ bistable circuits without the need for numerical simulation. We first consider two genetic toggle variants and two different positive feedback circuits, and show how specific topological properties present in each type of circuit can serve to increase the size of the regions of parameter space in which they function as switches. We then demonstrate that a single competitive monomeric activator added to a purely monomeric (and otherwise monostable) mutual repressor circuit is sufficient for bistability. Finally, we compare our approach with the Routh–Hurwitz method and derive consistent, yet more powerful, parametric conditions. The predictive power and ease of use of Sturm's theorem demonstrated in this work suggest that algebraic geometric techniques may be underused in biomolecular circuit analysis.nderused in biomolecular circuit analysis.)
Vehicle Motion Planning Using Stream Functions + (Borrowing a concept from hydrodynamic anal … Borrowing a concept from hydrodynamic analysis, this paper presents stream functions which satisfy Laplace's equation as a local-minima free method for producing potential-field based navigation functions in two dimensions. These functions generate smoother paths (i.e. more suited to aircraft-like vehicles) than previous methods. A method is developed for constructing analytic stream functions to produce arbitrary vehicle behaviors while avoiding obstacles, and an exact solution for the case of a single uniformly moving obstacle is presented. The effects of introducing multiple obstacles are discussed and current work in this direction is detailed. Experimental results generated on the Cornell RoboFlag testbed are presented and discussed.oFlag testbed are presented and discussed.)
Uniting local and global controllers for the Caltech ducted fan + (Caltech's ducted fan experiment is used as … Caltech's ducted fan experiment is usedas a case study to investigate the properties ofan algorithm for uniting local and global controllersproposed in (Teel and Kapoor, 1997)To simplify the control design process and to illustrate robustness,the ducted fan ismodeled as a linear system with input rate limits.The local controller is an (fairly aggressive) LQR state feedback whilethe (semi-)global controller is a much less aggressiveLQR state feedback. Closed-loop simulation resultsare providedusing a fully nonlinear model of the ducted fan derivedfrom wind tunnel data. Experimental results are alsoprovided using the actual Caltech ducted fan.vided using the actual Caltech ducted fan.)
A chemical reaction network model of PURE + (Cell-free expression systems provide a met … Cell-free expression systems provide a method for rapid DNA circuit prototyping and functional protein synthesis. While crude extracts remain a black box with many components carrying out unknown reactions, the PURE system contains only the required transcription and translation components for protein production. All proteins and small molecules are at known concentrations, opening up the possibility of detailed modeling for reliable computational predictions. However, there is little to no experimental data supporting the expression of target proteins for detailed protein models PURE models. In this work, we build a chemical reaction network transcription model for PURE protein synthesis. We compare the transcription models using DNA encoding for the malachite-green aptamer (MGapt) to measure mRNA production. Furthermore, we expand the PURE detailed translation model for an arbitrary set of amino acids and length. Lastly, we combine the transcription and the expanded translation models to create a PURE protein synthesis model built purely from mass-action reactions. We use the combined model to capture the translation of a plasmid encoding MGapt and deGFP under a T7-promoter and a strong RBS. The model accurately predicts the MGapt mRNA production for the first two hours, the dynamics of deGFP expression, and the total protein production with an accuracy within 10%.in production with an accuracy within 10%.)
Metabolic perturbations to an E. coli-based cell-free system reveal a trade-off between transcription and translation + (Cell-free transcription-translation (TX-TL … Cell-free transcription-translation (TX-TL) systems have been used for diverse applications, from prototyping gene circuits to providing a platform for the development of synthetic life, but their performance is limited by issues such as batch-to-batch variability, poor predictability, and limited lifetime. These issues stem largely from the fact that cell lysate contains an active and complex metabolism whose effect on TX-TL has remained largely uncharacterized.Motivated by a minimal model of cell-free metabolism, this work explored the effects of energy molecules, which power TX-TL, and fuel molecules, which regenerate energy by harnessing core metabolism, on an E. coli -based TX-TL system. This work reports a compensatory interaction between TX-TL components Mg2+ and 3-phosphoglyceric acid (3-PGA, used to regenerate ATP), where if one component’s concentration is increased, the other’s must likewise be increased to maintain optimal translation. Furthermore, maximum mRNA and protein production occur in opposite concentration regimes of Mg+2 and 3-PGA, suggesting a TX-TL trade-off. To explore the observed phenomenon, transcription and translation were decoupled. Under translation inhibition, transcriptional output was uniform across Mg2+ and 3-PGA concentrations, but in a translation-only system, maximum protein production occurred in the previously found optimal regime of Mg2+ and 3-PGA. Using alternative fuels to regenerate energy, this work found that the trade-off is universal across the different fuel sources, and that a system’s position along the trade-off is determined strongly by Mg2+. The location and slope of the trade-off curve are determined strongly by DNA concentration, cell lysate batch, and the fraction of cell lysate in a reaction. Finally, in systems where additional energy is supplied and where a fuel source is absent, the trade-off is absent.Overall, these results suggest the trade-off arises from limitations in translation regulation and efficient energy regeneration. This work represents a significant advancement in understanding the effects of fuel and energy metabolism on TX-TL in cell-free systems and lays the foundation for improving TX-TL performance, lifetime, standardization, and prediction.lifetime, standardization, and prediction.)
Proof of concept continuous event logging in living cells + (Cells must detect and respond to molecular … Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells don’t usually change their DNA in response to outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to small molecule stimuli. Known DNA sequences can be repeatedly integrated in a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore sequencing technology. by Oxford Nanopore sequencing technology.)
Feedback Stabilization of Steady-State and Hopf Bifurcations: the Multi-input Case + (Classification of stabilizability is obtai … Classification of stabilizability is obtained for multi-input nonlinear systemspossessing a simple steady-state or Hopf bifurcation with the critical mode being linearlyuncontrollable. Stabilizability is defined as the existence of a sufficiently smooth statefeedback such that the bifurcation for the closed loop system is supercritical, and in themeantime, the linearly controllable modes are locally asymptotically stable. Necessary andsufficient conditions of stabilizability are derived under certain nondegeneracyconditions. Explicit construction of stabilizing feedbacks is obtained for the cases whenthe system is stabilizable.the cases when the system is stabilizable.)
Multi-Hop Relay Protocols for Fast Consensus Seeking + (Consensus protocols in coordinated multi-a … Consensus protocols in coordinated multi-agent systems aredistributed algorithms. Just using local informationavailable to each single agent, all agents converge to anidentical consensus state and the convergence speed isdetermined by the algebraic connectivity of thecommunication network. In order to achieve a fasterconsensus seeking, we propose multi-hop relay protocolsbased on the current ``nearest neighbor rules'' consensusprotocols. By employing multiple-hop paths in the network,more information is passed around and each agent enlargesits "available" neighborhood. We demonstrate that theserelay protocols can increase the algebraic connectivitywithout physically adding or changing any communicationlinks. Moreover, time delay sensitivity of relay protocolsare discussed in detail. We point out that a trade offexists between convergence performance and time delayrobustness. Simulation results are also provided to verifythe efficiency of relay protocols. verify the efficiency of relay protocols.)
Pacti: Scaling Assume-Guarantee Reasoning for System Analysis and Design + (Contract-based design is a method to facil … Contract-based design is a method to facilitate modular system design. While there has been substantial progress on the theory of contracts, there has been less progress on scalable algorithms for the algebraic operations in this theory. In this paper, 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 then illustrate the use of Pacti in a variety of case studies.use of Pacti in a variety of case studies.)
Graph Laplacians and Stabilization of Vehicle Formations + (Control of vehicle formations has emerged … Control of vehicle formations has emerged as a topic of significant interest to the controlscommunity. In this paper, we merge tools from graph theory and control theory to derivestability criteria for formation stabilization. The interconnection between vehicles (i.e., whichvehicles are sensed by other vehicles) is modeled as a graph, and the eigenvalues of the Laplacianmatrix of the graph are used in stating a Nyquist-like stability criterion for vehicle formations.The location of the Laplacian eigenvalues can be correlated to the graph structure, and thereforeused to identify desirable and undesirable formation interconnection topologies.able formation interconnection topologies.)
Optimal and Cooperative Control of Vehicle Formations + (Control of vehicle formations has emerged … Control of vehicle formations has emerged as a topic of significant interest to the controls community. In applications such as microsatellites and underwater vehicles, formations have the potential for greater functionality and versatility than individual vehicles. In this thesis, we investigate two topics relevant to control of vehicle formations: optimal vehicle control and cooperative control.ehicle control and cooperative control.)
An Experimental Comparison of Tradeoffs in Using Compliant Manipulators for Robotic Grasping Tasks + (Controllers developed for control of flexi … Controllers developed for control of flexible-link robots in hybridforce-position control tasks by a new singular perturbation analysisof flexible manipulators are implemented on an experimental two-robotgrasping setup. Various performance criteria are set up andexperimental results are discussed within that setting to showtradeoffs in using flexible link robots for grasping. We conclude thatlarge flexibility can be controlled without too much additionaleffort, has performance comparable to rigid robots and possessesenhancing properties which make it attractive for use in certain typesof applications. for use in certain types of applications.)
Robust Estimation Framework with Semantic Measurements + (Conventional simultaneous localization and … Conventional simultaneous localization and map- ping (SLAM) algorithms rely on geometric measurements and require loop-closure detections to correct for drift accumulated over a vehicle trajectory. Semantic measurements can add measurement redundancy and an alternative form of loop closure. We propose two different estimation algorithms that incorporate semantic measurements provided by vision-based object classifiers. An a priori map of regions where the objects can be detected is assumed. The first estimation framework is posed as a maximum-likelihood problem, where the likelihood function for semantic measurements is derived from the con- fusion matrices of the object classifiers. The second estimation framework is comprised of two parts: 1) a continuous-state estimation formulation that includes semantic measurements as a form of state constraints and 2) a discrete-state estimation formulation used to compute the certainty of object detection measurements using a Hidden Markov Model (HMM). The advantages of incorporating semantic measurements in these frameworks are demonstrated in numerical simulations. In particular, the proposed estimation algorithms improve upon the robustness and accuracy of conventional SLAM algorithms. Also, the certainty metric of object detection measurements derived from the HMM in our simulation are greater than the certainty levels provided by the confusion matrix in object classification algorithms.atrix in object classification algorithms.)
Convergence Properties of Dynamic Agents Consensus Networks with Broken Links + (Convergence properties of distributed cons … Convergence properties of distributed consensus protocols on networks of dynamical agents have been analyzed by combinations of algebraic graph theory and control theory tools under certain assumptions, such as strong connectivity. Strong connectivity can be regarded as the requirement that the information of each agent propagates to all the others, possibly with intermediate steps and manipulations. However, because of network failures or malicious attacks, it is possible that this assumption no longer holds, so that some agents are only receiving or only transmitting information from other subsets of agents. In this case, strong connectivity is replaced by weak connectivity. We analyze the convergence properties of distributed consensus on directed graphs with weakly connected components. We show conditions for which the agreement is reached, and, for the cases in which such conditions do not hold, we provide bounds on the residual disagreement. in terms of the number of agents that must fail for thehe number of agents that must fail for the)
Variable Elimination for Scalable Receding Horizon Temporal Logic Planning + (Correct-by-construction synthesis of high- … Correct-by-construction synthesis of high-level re- active control relies on the use of formal methods to generate controllers with provable guarantees on their behavior. While this approach has been successfully applied to a wide range of systems and environments, it scales poorly with the increasing size of the environment. A receding horizon framework was recently proposed to mitigate this computational blowup, by decomposing the global control problem into several tractable subproblems. The existence of a global controller is ensured through symbolic checks of the specification, and local controllers are synthesized when needed, using the current state of the environment as the initial condition. This reduces the size of the synthesized strategy, but does not provide much improvement for problems with large dynamic environments, because the large number of possible global environment strategies. Ad hoc methods to locally restrict the environment have previously been used, at the risk of losing correctness. This paper presents a method of reducing specifications by eliminating locally redundant variables, while maintaining the correctness of controllers. We demonstrate the method using an autonomous car example, on problem sizes that were previously unsolvable due to the number of variables in the environment. We also demonstrate how the reduced specifications can be used to identify opportunities for reusing the synthesized local controllers.reusing the synthesized local controllers.)
Quantification and Minimization of Crosstalk Sensitivity in Networks + (Crosstalk is defined as the set of unwante … Crosstalk is defined as the set of unwanted interactions among the different entities of a network. Crosstalk is present in various degrees in every system where information is transmitted through a means that is accessible by all the individual units of the network. Using concepts from graph theory, we introduce a quantifiable measure for sensitivity to crosstalk, and analytically derive the structure of the networks in which it is minimized. It is shown that networks with an inhomogeneous degree distribution are more robust to crosstalk than corresponding homogeneous networks. We provide a method to construct the graph with the minimum possible sensitivity to crosstalk, given its order and size. Finally, for networks with a fixed degree sequence, we present an algorithm to find the optimal interconnection structure among their vertices.connection structure among their vertices.)
Design and application of stationary phase combinatorial promoters + (Current bacterial synthetic circuits rely … Current bacterial synthetic circuits rely on the fast dilution and high protein expression that occurs during exponential phase. However, constant exponential phase is both difficult to ensure in a lab environment and almost certainly impractical in any natural setting. Here, we characterize the performance of 13 E. coli native 38 promoters, as well as a previously identified 38 consensus promoter. We then make tetO combinatorial versions of the three strongest promoters to allow for inducible delayed expression. The design of these combinatorial promoters allows for design of circuits with inducible stationary phase activity that can be used for phase-dependent delays in dynamic circuits or spatial partitioning of biofilms.cuits or spatial partitioning of biofilms.)
System-level studies of a cell-free transcription-translation platform for metabolic engineering + (Current methods for assembling biosyntheti … Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.tly applied to in vivo strain development.)
Prototyping 1,4-butanediol (BDO) biosynthesis pathway in a cell-free transcription-translation (TX-TL) system + (Current methods for assembling metabolic p … Current methods for assembling metabolic pathways require a process of repeated trial and error and have a long design-build-test cycle. Further, it remains a challenge to precisely tune enzyme expression levels for maximizing target metabolite production. Recently it was shown that a cell-free transcriptional-translation system (TX-TL) can be used to rapidly prototype novel complex biocircuits as well as metabolic pathways. TX-TL systems allow protein expression from multiple DNA pieces, opening up the possibility of modulating concentrations of DNA encoding individual pathway enzymes and testing the related effect on metabolite production. In this work, we demonstrate TX-TL as a platform for exploring the design space of metabolic pathways using a 1,4-BDO biosynthesis pathway as an example. Using TX-TL, we verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as a limiting step of the 1,4-BDO pathway. We further tested combinations of various enzyme expression levels and found increasing downstream enzyme expression levels improved 1,4-BDO production.ession levels improved 1,4-BDO production.)
TRILL: Orchestrating Modular Deep-Learning Workflows for Democratized, Scalable Protein Analysis and Engineering + (Deep-learning models have been rapidly ado … Deep-learning models have been rapidly adopted by many fields, partly due to the deluge of data humanity has amassed. In particular, the petabases of biological sequencing data enable the unsupervised training of protein language models that learn the “language of life.” However, due to their prohibitive size and complexity, contemporary deep-learning models are often unwieldy, especially for scientists with limited machine learning backgrounds. TRILL (TRaining and Inference using the Language of Life) is a platform for creative protein design and discovery. Leveraging several state-of-the-art models such as ESM-2, DiffDock, and RFDiffusion, TRILL allows researchers to generate novel proteins, predict 3-D structures, extract high-dimensional representations of proteins, functionally classify proteins and more. What sets TRILL apart is its ability to enable complex pipelines by chaining together models and effectively merging the capabilities of different models to achieve a sum greater than its individual parts. Whether using Google Colab with one GPU or a supercomputer with hundreds, TRILL allows scientists to effectively utilize models with millions to billions of parameters by using optimized training strategies such as ZeRO-Offload and distributed data parallel. Therefore, TRILL not only bridges the gap between complex deep-learning models and their practical application in the field of biology, but also simplifies the orchestration of these models into comprehensive workflows, democratizing access to powerful methods. democratizing access to powerful methods.)
The effects of time-varying temperature on delays in genetic networks + (Delays in gene networks result from the se … Delays in gene networks result from the sequential nature of protein assembly. However, it is unclear how models of gene networks that use delays should be modified when considering time-dependent changes in temperature. This is important, as delay is often used in models of genetic oscillators that can be entrained by periodic fluctuations in temperature. Here, we analytically derive the time dependence of delay distributions in response to time-varying temperature changes. We find that the resulting time-varying delay is nonlinearly dependent on parameters of the time-varying temperature such as amplitude and frequency, therefore, applying an Arrhenius scaling may result in erroneous conclusions. We use these results to examine a model of a synthetic gene oscillator with temperature compensation. We show that temperature entrainment follows from the same mechanism that results in temperature compensation. Under a common Arrhenius scaling alone, the frequency of the oscillator is sensitive to changes in the mean temperature but robust to changes in the frequency of a periodically time-varying temperature. When a mechanism for temperature compensation is included in the model, however, we show that the oscillator is entrained by periodically varying temperature even when maintaining insensitivity to the mean temperature.ing insensitivity to the mean temperature.)
Population diversification in a yeast metabolic program promotes anticipation of environmental shifts + (Delineating the strategies by which cells … Delineating the strategies by which cells contend with combinatorial changing environments is crucial for understanding cellular regulatory organization. When presented with two carbon sources, microorganisms first consume the carbon substrate that supports the highest growth rate (e.g. glucose) and then switch to the secondary carbon source (e.g. galactose), a paradigm known as the Monod model. Sequential sugar utilization has been attributed to transcriptional repression of the secondary metabolic pathway, followed by activation of this pathway upon depletion of the preferred carbon source. In this work, we challenge this notion. Although Saccharomyces cerevisiae cells consume glucose before galactose, we demonstrate that the galactose regulatory pathway is activated in a fraction of the cell population hours before glucose is fully consumed. This early activation reduces the time required for the population to transition between the two metabolic programs and provides a fitness advantage that might be crucial in competitive environments. Importantly, these findings define a new paradigm for the response of microbial populations to combinatorial carbon sources.pulations to combinatorial carbon sources.)
Context-Aided Variable Elimination for Requirement Engineering + (Deriving system-level specifications from … Deriving system-level specifications from component specifications usually involves the elimination of variables that are not part of the interface of the top-level system. This paper presents algorithms for eliminating variables from formulas by computing refinements or relaxations of these formulas in a context. We discuss a connection between this problem and optimization and give efficient algorithms to compute refinements and relaxations of linear inequality constraints.axations of linear inequality constraints.)

(Deriving system-level specifications from component specifications usual)

Analysis of Circuits for Dosage Control in Microbial Populations + (Designing genetic circuits to control the … Designing genetic circuits to control the behaviors of microbial populations is an ongoing challenge in synthetic biology. Here we analyze circuits which implement dosage control by controlling levels of a global signal in a microbial population in face of varying cell density, growth rate, and environmental dilution. We utilize the Lux quorum sensing system to implement dosage control circuits, and we analyze the dynamics of circuits using both simplified analytical analysis and in silico simulations. We demonstrate that strong negative feedback through inhibiting LuxI synthase expression along with AiiA degradase activity results in circuits with fast response times and robustness to cell density and dilution rate. We find that degradase activity yields robustness to variations in population density for large population sizes, while negative feedback to synthase production decreases sensitivity to dilution rates.n decreases sensitivity to dilution rates.)
Cooperation Enhances Robustness of Coexistence in Spatially Structured Consortia + (Designing synthetic microbial consortia is … Designing synthetic microbial consortia is an emerging area in synthetic biology and a major goal is to realize stable and robust coexistence of multiple species. Cooperation and competition are fundamental intra/interspecies interactions that shape population level behaviors, yet it is not well-understood how these interactions affect the stability and robustness of coexistence. In this paper, we show that communities with cooperative interactions are more robust to population disturbance, e.g., depletion by antibiotics, by forming intermixed spatial patterns. Meanwhile, competition leads to population spatial heterogeneity and more fragile coexistence in communities. Using reaction-diffusion and nonlocal PDE models and simulations of a two-species E. coli consortium, we demonstrate that cooperation is more beneficial than competition in maintaining coexistence in spatially structured consortia, but not in well-mixed environments. This also suggests a trade-off between constructing heterogeneous communities with localized functions and maintaining robust coexistence. The results provide general strategies for engineering spatially structured consortia by designing interspecies interactions and suggest the importance of cooperation for biodiversity in microbial community.n for biodiversity in microbial community.)
Flow-Based Synthesis of Reactive Tests for Discrete Decision-Making Systems with Temporal Logic Specifications + (Designing tests to evaluate if a given aut … 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.al robots for a variety of specifications.)
On-chip membrane protein cell-free expression enables development of a direct binding assay: A curious case of potassium channel KcsA-Kv1.3 + (Despite the significant role integral memb … Despite the significant role integral membrane proteins (IMPs) play in the drug discovery process, it remains extremely challenging to express, purify, and in vitro stabilize them for detailed biophysical analyses. Cell-free transcription-translation systems have emerged as a promising alternative for producing complex proteins, but they are still not a viable option for expressing IMPs due to improper post-translational folding of these proteins. We have studied key factors influencing in vitro folding of cell-free-expressed IMPs, particularly oligomeric proteins (i.e., ion channels). Using a chimeric ion channel, KcsA-Kv1.3 (K-K), as a model IMP, we have investigated several physiochemical determinants including artificial bilayer environments (i.e., lipid, detergent) for K-K in vitro stabilization. We observed that fusion of a ‘superfolder’ green fluorescent protein (sfGFP) to K-K as a protein expression reporter not only improves the protein yield, but surprisingly facilitates the K-K tetramer formation, probably by enhancing the solubility of monomeric K-K. Additionally, anionic lipids (i.e., DMPG) were found to be essential for the correct folding of cell-free-expressed monomeric K-K into tetramer, underscoring the importance of lipid-protein interaction in maintaining structural-functional integrity of ion channels. We further developed methods to integrate cell-free-expressed IMPs directly onto a biosensor chip. We employed a solid-supported lipid bilayer onto the surface plasmon resonance (SPR) chip to insert nascent K-K in a membrane. In a different approach, an anti-GFP-functionalized surface was used to capture in situ expressed K-K via its sfGFP tag. Interestingly, only the K-K-functionalized capture surface prepared by the latter strategy was able to interact with K-K's small binding partners. This generalizable approach can be further extended to other membrane proteins for developing direct binding assays involving small ligands.ct binding assays involving small ligands.)
Optimal nonlinear guidance with inner-loop feedback for hypersonic re-entry + (Development of feasible G&C (guidance … Development of feasible G&C (guidance and control) methods for precision atmospheric re-entry has remained a challenge since pre-Apollo-era space exploration. The inherent difficulty arises from the governing hypersonic dynamics being significantly nonlinear, subject to parametric uncertainty, and limited with control authority. Vehicle safety requirements impose further constraints, and desired cost objectives complicate an already difficult G&C problem. The scope of this paper is to present a guidance algorithm for optimal trajectory generation based on a reduced-dimension reentry formulation. Preliminary simulations demonstrate the algorithm with feedback used to track the guidance trajectory in the presence of initial state uncertainty. The objective is to further this approach toward an onboard receding-horizon implementation toward an onboard receding-horizon implementation)
Geometric Phases, Control Theory, and Robotics + (Differential geometry and nonlinear contro … Differential geometry and nonlinear control theory provide essentialtools for studying motion generation in robot systems. Two areaswhere progress is being made are motion planning for mobile robots onthe factory floor (or on the surface of Mars), and control of highlyarticulated robots---such as multifingered robot hands and robot``snakes''---for medical inspection and manipulation inside thegastrointestinal tract. A common feature of these systems is the roleof constraints on the behavior of the system. Typically, theseconstraints force the instantaneous velocities of the system to lie ina restricted set of directions, but do not actually restrict thereachable configurations of the system. A familiar example in whichthis geometric structure can be exploited is parallel parking of anautomobile, where periodic motion in the driving speed and steeringangle can be used to achieve a net sideways motion. By studying thegeometric nature of velocity constraints in a more general setting, itis possible to synthesize gaits for snake-like robots, generateparking and docking maneuvers for automated vehicles, and study theeffects of rolling contacts on multifingered robot hands. As inparallel parking, rectification of periodic motions in the controlvariables plays a central role in the techniques which are used togeneration motion in this broad class of robot systems.tion in this broad class of robot systems.)
Differentially Flat Nonlinear Control Systems + (Differentially flat systems are underdeter … Differentially flat systems are underdetermined systems of (nonlinear) ordinary differential equations(ODEs) whose solution curves are in smooth one-one correspondence with arbitrary curves in a spacewhose dimension equals the number of equations by which the system is underdetermined. For controlsystems this is the same as the number of inputs. The components of the map from the system space tothe smaller dimensional space are referred to as the flat outputs. Flatness allows one to systematicallygenerate feasible trajectories in a relatively simple way. Typically the flat outputs may depend on theoriginal independent and dependent variables in terms of which the ODEs are written as well as finitelymany derivatives of the dependent variables. Flatness of systems underdetermined by one equation iscompletely characterised by Elie Cartan's work. But for general underdetermined systems no completecharacterisation of flatness exists. In this dissertation we describe two different geometric frameworks for studying flatness and provideconstructive methods for deciding the flatness of certain classes of nonlinear systems and for findingthese flat outputs if they exist. We first introduce the concept of ``absolute equivalence'' due to Cartanand define flatness in this frame work. We provide a method of testing for the flatness of systems, whichinvolves making a guess for all but one of the flat outputs after which the problem is reduced to the casesolved by Cartan. Secondly we present an alternative geometric approach to flatness which uses ``jetbundles'' and present a theorem which partially characterises flat outputs that depend only on theoriginal variables but not on their derivatives, for the case of systems described by two independentone-forms in arbitrary number of variables. Finally, for the class of Lagrangian mechanical systems whosenumber of control inputs is one less than the number of degrees of freedom, we provide acharacterisation of flat outputs that depend only on the configuration variables, but not on theirderivatives. This characterisation makes use of the Riemannian metric provided by the kinetic energy ofthe system.ic provided by the kinetic energy of the system.)
Asynchronous Distributed Averaging on Communication Networks + (Distributed algorithms for averaging have attracted interest in the control and sensing literature. However, previous works have not addressed some practical concerns that will arise in actual implementations on packet-switched communication networks.....)
Distributed Averaging on Asynchronous Communication Networks + (Distributed algorithms for averaging have … Distributed algorithms for averaging have at- tracted interest in the control and sensing literature. However, previous works have not addressed some practical concerns that will arise in actual implementations on packet-switched communication networks such as the Internet. In this paper, we present several implementable algorithms that are robust to asynchronism and dynamic topology changes. The algorithms do not require global coordination and can be proven to converge under very general asynchronous timing assumptions. Our results are verified by both simulation and experiments on a real-world TCP/IP network.periments on a real-world TCP/IP network.)
A Model-Free Algorithm for Extremely Resilient Navigation + (Due to the increasing complexity of space … Due to the increasing complexity of space missions and distance to exploration targets, future robotic systems used for space exploration call for more resilience and autonomy. Instead of minimizing the failure risk, we are focusing on missions that will inevitably encounter significant failures and are developing an algorithm that will autonomously reconfigure the system controller to continue to make progress towards the mission goal despite being in a reduced capacity state - we call this extreme resilience. In this paper, we develop a model-free framework to autonomously react to locomotion failures of robotic systems. This is done by the use of a neural network for path planning using the neuroevolution of aug- menting topologies (NEAT) algorithm and a dynamic database of possible moves and their effect on the system’s position and orientation. Two modes of failure detection and resolution are being introduced: (a) relative position failure detection, which is triggered by large, unexpected moves and results in a complete update of the database before a retraining of the neural network, and (b) absolute position failure detection, which triggers from large build-ups of position error from small failures and will induce a retraining of the neural network without an explicit database reset. We implement and validate this framework on a high-fidelity planetary rover simulation using Unreal Engine and on a hardware setup of a TurtleBot2 with a PhantomX Pincher robot arm.tleBot2 with a PhantomX Pincher robot arm.)
A population-based temporal logic gate for timing and recording chemical events + (Engineered bacterial sensors have potentia … Engineered bacterial sensors have potential applications in human health monitoring, environmental chemical detection, and materials biosynthesis. While such bacterial devices have long been engineered to differentiate between combinations of inputs, their potential to process signal timing and duration has been overlooked. In this work, we present a two-input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. Our temporal logic gate design relies on unidirectional DNA recombination mediated by bacteriophage integrases to detect and encode sequences of input events. For an E. coli strain engineered to contain our temporal logic gate, we compare predictions of Markov model simulations with laboratory measurements of final population distributions for both step and pulse inputs. Although single cells were engineered to have digital outputs, stochastic noise created heterogeneous single-cell responses that translated into analog population responses. Furthermore, when single-cell genetic states were aggregated into population-level distributions, these distributions contained unique information not encoded in individual cells. Thus, final differentiated sub-populations could be used to deduce order, timing, and duration of transient chemical events.and duration of transient chemical events.)
Addressable and adaptable intercellular communication via DNA messaging + (Engineered consortia are a major research … Engineered consortia are a major research focus for synthetic biologists because they can implement sophisticated behaviors inaccessible to single-strain systems. However, this functional capacity is constrained by their constituent strains’ ability to engage in complex communication. DNA messaging, by enabling information-rich channel-decoupled communication, is a promising candidate architecture for implementing complex communication. But its major advantage, its messages’ dynamic mutability, is still unexplored. We develop a framework for addressable and adaptable DNA messaging that leverages all three of these advantages and implement it using plasmid conjugation in E. coli. Our system can bias the transfer of messages to targeted receiver strains by 100- to 1000-fold, and their recipient lists can be dynamically updated in situ to control the flow of information through the population. This work lays the foundation for future developments that further utilize the unique advantages of DNA messaging to engineer previously-inaccessible levels of complexity into biological systems.els of complexity into biological systems.)
Role of interaction network topology in controlling microbial population in consortia + (Engineering microbial consortia is an impo … Engineering microbial consortia is an important new frontier for synthetic biology given its efficiency in performing complex tasks and endurance to environmental uncertainty. Most synthetic circuits regulate population level behaviors via cell-to-cell communications, which are affected by spatially heterogeneous environments. Therefore, it is important to understand the limits on controlling system dynamics that are determined by interconnections among cell agents and provide a control strategy for engineering consortia. Here, we build a network model for a fractional population control circuit in two-strain consortia, and characterize the cell-to-cell communication network by topological properties, such as symmetry, locality and connectivity. Using linear network control theory, we relate the network topology to system output tracking performance. We analytically and numerically demonstrate that the minimum network control energy for accurate tracking depends on locality difference between two cell populations and how strongly the controller node contributes to communication strength. To realize robust consortia, we can manipulate the communication network topology and construct strongly connected consortia by altering chemicals in environments. Our results ground the expected cell population dynamics in its spatially organized communication network, and inspire directions in cooperative control in microbial consortia.ooperative control in microbial consortia.)
Invariant Sets for Integrators and Quadrotor Obstacle Avoidance + (Ensuring safety through set invariance has … Ensuring safety through set invariance has proven a useful method in a variety of applications in robotics and control. However, finding analytical expressions for maximal invariant sets, so as to maximize the operational freedom of the system without compromising safety, is notoriously difficult for high-dimensional systems with input constraints. Here we present a generic method for characterizing invariant sets of nth-order integrator systems, based on analyzing roots of univariate polynomials. Additionally, we obtain analytical expressions for the orders n <= 4. Using differential flatness we subsequently leverage the results for the n = 4 case to the problem of obstacle avoidance for quadrotor UAVs. The resulting controller has a light computational footprint that showcases the power of finding analytical expressions for control-invariant sets.tical expressions for control-invariant sets.)
Construction of an inducible amyloid expression circuit in Bacillus megaterium: A case study with CsgA and TasA + (Environmental applications of synthetic bi … Environmental applications of synthetic biology such as water remediation require engineered strains to function robustly in a fluctuating and potentially hostile environment. The construction of synthetic biofilm formation circuits could potentially alleviate this issue by promoting cell survival. Towards this end, we construct a xylose-inducible system for the expression of the functional amyloids CsgA and TasA in the soil bacterium Bacillus megaterium. We find that although both amyloids are expressed, only TasA is successfully exported from the cells. Furthermore, expression of CsgA results in a significant growth penalty for the cells while expression of TasA does not. Finally, we show that TasA expression conveys a small but detectable increase in cells’ adhesion to nickel beads. These results suggest that TasA is a promising candidate for future work on synthetic biofilm formation in B. megaterium.thetic biofilm formation in B. megaterium.)
Performance Metrics for a Biomolecular Step Response + (Establishing performance metrics is a key … Establishing performance metrics is a key part of a systematic design process. In particular, specifying metrics useful for quantifying performance in the ongoing efforts towards biomolecular circuit design is an important problem. Here we address this issue for the design of a fast biomolec- ular step response that is uniform across different cells and widely different environmental conditions using a combination of simple mathematical models and experimental measure- ments using single-cell time-lapse microscopy. We evaluate two metrics, the difference of the step response from an ideal step and the relative difference between multiple realizations of the step response, that can provide a single number to measure performance. We use a model of protein production- degradation to show that these performance metrics correlate with response features of speed and noise. Finally, we work through an experimental methodology to estimate these metrics for step responses that have been acquired for inducible protein expression circuits in E. coli. These metrics will be useful to evaluate biomolecular step responses, as well as for setting similar performance measures for other design goals.rformance measures for other design goals.)
An Experimental Comparison of Controllers for a Vectored Thrust, Ducted Fan Engine + (Experimental comparisons between four diff … Experimental comparisons between four different control designmethodologies are applied to a small vectored thrust engine.Each controller is applied to three trajectories of varyingaggressiveness. The control strategies considered are LQR,H_infty, gain scheduling, and feedback linearization. The experiments show that gain schedulingis essential to achieving good performance. Thestrengths and weaknesses of each methodology are also examined.ses of each methodology are also examined.)
Model-Based Control of Cavity Oscillations, Part II: System Identificaion and Analysis + (Experiments using active control to reduce … Experiments using active control to reduce oscillationsin the flow past a rectangular cavity have uncoveredsurprising phenomena: in the controlled system,often new frequencies of oscillation appear, andoften the main frequency of oscillation is split intotwo sideband frequencies. The goal of this paper isto explain these effects using physics-based models,and to use these ideas to guide control design.We present a linear model for the cavity flow,based on the physical mechanisms of the familiarRossiter model. Experimental data indicatesthat under many operating conditions, the oscillationsare not self-sustained, but in fact are causedby amplification of external disturbances. Wepresent some experimental results demonstratingthe peak-splitting phenomena mentioned above, usethe physics-based model to study the phenomena,and discuss fundamental performance limitationswhich limit the achievable performance of any controlscheme.ievable performance of any control scheme.)
Planning and Optimization for Multi-Robot Planetary Cave Exploration under Intermittent Connectivity Constraints + (Exploring subsurface structures with auton … Exploring subsurface structures with autonomous robots is of growing interest in the context of planetary caves studies. Communication between robots in these environments is severely degraded which complicates coordination and information distribution. In this paper we focus on planning for mobility and communication in a cave exploration scenario where the situational awareness of a static base station is critical. We propose a notion of information-consistency where a plan itself is part of the information to be shared between robots, and propose a method for generating informationconsistent plans. We discuss in detail how the resulting plan can be robustly implemented with minimal communication through local mission executives that run on individual robots. We describe preliminary results on the performance of the planning algorithm and integration of the local mission executives in a high-fidelity simulation environment.in a high-fidelity simulation environment.)
Failure Probability of Verifiable Goal-based Control Programs due to State Estimation Uncertainty + (Fault tolerance and safety verification of … Fault tolerance and safety verification of control systems that have state estimation uncertainty are essential for the success of autonomous robotic systems. A software control architecture called Mission Data System, developed at the Jet Propulsion Laboratory, uses goal networks as the control program for autonomous systems. Certain types of goal networks can be converted into linear hybrid systems and verified for safety using existing symbolic model checking software. A process for calculating the probability of failure of some verifiable goal networks due to state estimation uncertainty is presented. Extensions of this procedure to include other types of uncertainties are discussed, and example problems are presented to illustrate these procedures. presented to illustrate these procedures.)
Control Program Verification for a Sample Titan Aerobot Mission + (Fault tolerance and safety verification of … Fault tolerance and safety verification of control systems are essential for the success of autonomous robotic systems. A control architecture called Mission Data System (MDS), developed at the Jet Propulsion Laboratory, takes a goal-based control approach. A software algorithm for converting goal network control programs into linear hybrid systems exists and is a bisimulation; the resulting linear hybrid system can be verified for safety in the presence of failures using existing symbolic model checkers, and thus the original goal network is verified. A substantial example control program based on a proposed mission to Titan, a moon of Saturn, is converted using the procedures discussed. converted using the procedures discussed.)
Automatic Conversion Software for the Safety Verification of Goal-Based Control Programs + (Fault tolerance and safety verification of … Fault tolerance and safety verification of control systems are essential for the success of autonomous robotic systems. A control architecture called Mission Data System (MDS), developed at the Jet Propulsion Laboratory, takes a goal-based control approach. In this paper, a software algorithm for converting goal network control programs into linear hybrid systems is described. The conversion process is a bisimulation; the resulting linear hybrid system can be verified for safety in the presence of failures using existing symbolic model checkers, and thus the original goal network is verified. A moderately complex goal network control program is converted to a linear hybrid system using the automatic conversion software and then verified.tic conversion software and then verified.)
Conversion and Verification Procedure for Goal-Based Control Programs + (Fault tolerance and safety verification of … Fault tolerance and safety verification of control systems are essential for the success of autonomous robotic systems. A control architecture called Mission Data System, developed at the Jet Propulsion Laboratory, takes a goal-based control approach. In this paper, a method for converting goal network control programs into linear hybrid systems is developed. The linear hybrid system can then be verified for safety in the presence of failures using existing symbolic model checkers. An example task is developed and successfully verified using HyTech, a symbolic model checking software for linear hybrid systems.ecking software for linear hybrid systems.)
Safety Verification of Fault Tolerant Goal-based Control Programs with Estimation Uncertainty + (Fault tolerance and safety verification of … Fault tolerance and safety verification of control systems that have state variable estimation uncertainty are essential for the success of autonomous robotic systems. A software control architecture called Mission Data System, developed at the Jet Propulsion Laboratory, uses goal networks as the control program for autonomous systems. Certain types of goal networks can be converted into linear hybrid systems and verified for safety using existing symbolic model checking software. A process for calculating the probability of failure of certain classes of verifiable goal networks due to state estimation uncertainty is presented. A verifiable example task is presented and the failure probability of the control program based on estimation uncertainty is found.based on estimation uncertainty is found.)
Design of a genetic layered feedback controller in synthetic biological circuitry + (Feedback control is the key to achieve rob … Feedback control is the key to achieve robust performances for many engineered systems. However, its application in biological contexts is still largely unexplored. In this work, we designed, analyzed and simulated a layered controller functioning at both molecular and populational levels. First, we used a minimal model of three states to represent a system where state A activates state B; state R is a by-product of state B that acts as a negative feedback regulating both state A, B, and sequentially R. We call the feedback applied to state B a cis feedback and the one applied to state A a trans feedback. Through stability analysis via linearization at equilibrium and sensitivity analysis at transient state, we found that the cis feedback attenuates disturbances better but recovers slower; the trans feedback recovers faster but has more dramatic responses to fluctuations; the layered feedback demonstrates both advantageous traits of the two single layers. Then we designed two versions of synthetic genetic circuits to implement the layered controller in living cells. One version with an sRNA as regulator R, the other with a transcription factor protein as the regulator R. The analysis and dynamical simulation of the models confirmed the analytical results from the minimal model. At the same time, we found that the protein regulated feedback controls have faster recovery speed but the RNA version has a stronger disturbance attenuation effect.a stronger disturbance attenuation effect.)

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