605 research outputs found

    Synthetic in vitro transcriptional oscillators

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    The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells

    Bistability of an In Vitro Synthetic Autoregulatory Switch

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    The construction of synthetic biochemical circuits is an essential step for developing quantitative understanding of information processing in natural organisms. Here, we report construction and analysis of an in vitro circuit with positive autoregulation that consists of just four synthetic DNA strands and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template allowed a rational design of a synthetic DNA switch regulated by its RNA output acting as a transcription activator. We verified that the thermodynamic and kinetic constraints dictated by the sequence design criteria were enough to experimentally achieve the intended dynamics: a transcription activator configured to regulate its own production. Although only RNase H is necessary to achieve bistability of switch states, RNase R is necessary to maintain stable RNA signal levels and to control incomplete degradation products. A simple mathematical model was used to fit ensemble parameters for the training set of experimental results and was then directly applied to predict time-courses of switch dynamics and sensitivity to parameter variations with reasonable agreement. The positive autoregulation switches can be used to provide constant input signals and store outputs of biochemical networks and are potentially useful for chemical control applications

    Ensemble Bayesian Analysis of Bistability in a Synthetic Transcriptional Switch

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    An overarching goal of synthetic and systems biology is to engineer and understand complex biochemical systems by rationally designing and analyzing their basic component interactions. Practically, the extent to which such reductionist approaches can be applied is unclear especially as the complexity of the system increases. Toward gradually increasing the complexity of systematically engineered systems, programmable synthetic circuits operating in cell-free in vitro environments offer a valuable testing ground for principles for the design, characterization, and analysis of complex biochemical systems. Here we illustrate this approach using in vitro transcriptional circuits (“genelets”) while developing an activatable transcriptional switch motif and configuring it as a bistable autoregulatory circuit, using just four synthetic DNA strands and three essential enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease H, and ribonuclease R. Fulfilling the promise of predictable system design, the thermodynamic and kinetic constraints prescribed at the sequence level were enough to experimentally demonstrate intended bistable dynamics for the synthetic autoregulatory switch. A simple mathematical model was constructed based on the mechanistic understanding of elementary reactions, and a Monte Carlo Bayesian inference approach was employed to find parameter sets compatible with a training set of experimental results; this ensemble of parameter sets was then used to predict a test set of additional experiments with reasonable agreement and to provide a rigorous basis for confidence in the mechanistic model. Our work demonstrates that programmable in vitro biochemical circuits can serve as a testing ground for evaluating methods for the design and analysis of more complex biochemical systems such as living cells

    Neural network computation by in vitro transcriptional circuits

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    The structural similarity of neural networks and genetic regulatory networks to digital circuits, and hence to each other, was noted from the very beginning of their study [1, 2]. In this work, we propose a simple biochemical system whose architecture mimics that of genetic regulation and whose components allow for in vitro implementation of arbitrary circuits. We use only two enzymes in addition to DNA and RNA molecules: RNA polymerase (RNAP) and ribonuclease (RNase). We develop a rate equation for in vitro transcriptional networks, and derive a correspondence with general neural network rate equations [3]. As proof-of-principle demonstrations, an associative memory task and a feedforward network computation are shown by simulation. A difference between the neural network and biochemical models is also highlighted: global coupling of rate equations through enzyme saturation can lead to global feedback regulation, thus allowing a simple network without explicit mutual inhibition to perform the winner-take-all computation. Thus, the full complexity of the cell is not necessary for biochemical computation: a wide range of functional behaviors can be achieved with a small set of biochemical components

    Robust mixing in self-consistent linearized augmented planewave calculations

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    We devise a mixing algorithm for full-potential (FP) all-electron calculations in the linearized augmented planewave (LAPW) method. Pulay's direct inversion in the iterative subspace is complemented with the Kerker preconditioner and further improvements to achieve smooth convergence, avoiding charge sloshing and noise in the exchange-correlation potential. As the Kerker preconditioner was originally designed for the planewave basis, we have adapted it to the FP-LAPW method and implemented in the exciting code. Applications to the 2×22\times 2 Au(111) surface with a vacancy and to the Pd(111) surface demonstrate that this approach and our implementation work reliably with both density and potential mixing

    Adding Small Differences can Increase Similarity and Choice

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    Similarity plays a critical role in many judgments and choices. Traditional models of similarity posit that increasing the number of differences between objects cannot increase judged similarity between them. In contrast to these previous models, the present research shows that introducing a small difference in an attribute that previously was identical across objects can increase perceived similarity between those objects. We propose an explanation based on the idea that small differences draw more attention than identical attributes do and that people’s perceptions of similarity involve averaging attributes that are salient. We provide evidence that introducing small differences between objects increases perceived similarity. We also show that an increase in similarity decreases the difficulty of choice and the likelihood that a choice will be deferred. </jats:p

    Learning Rotation-Equivariant Features for Visual Correspondence

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    Extracting discriminative local features that are invariant to imaging variations is an integral part of establishing correspondences between images. In this work, we introduce a self-supervised learning framework to extract discriminative rotation-invariant descriptors using group-equivariant CNNs. Thanks to employing group-equivariant CNNs, our method effectively learns to obtain rotation-equivariant features and their orientations explicitly, without having to perform sophisticated data augmentations. The resultant features and their orientations are further processed by group aligning, a novel invariant mapping technique that shifts the group-equivariant features by their orientations along the group dimension. Our group aligning technique achieves rotation-invariance without any collapse of the group dimension and thus eschews loss of discriminability. The proposed method is trained end-to-end in a self-supervised manner, where we use an orientation alignment loss for the orientation estimation and a contrastive descriptor loss for robust local descriptors to geometric/photometric variations. Our method demonstrates state-of-the-art matching accuracy among existing rotation-invariant descriptors under varying rotation and also shows competitive results when transferred to the task of keypoint matching and camera pose estimation.Comment: Accepted to CVPR 2023, Project webpage at http://cvlab.postech.ac.kr/research/REL