Copy number variation is now recognized as one of the major sources of genetic variation among individuals in natural populations of any species. However, the relevance of these unexpected ...observations goes beyond diagnosing high diversity.
Here, it is argued that the molecular rates of copy number variation, mainly the deletion rate upon variation, determine the evolutionary road of the genome regarding size. Genetic drift will govern this process only if the effective population size is lower than the inverse of the deletion rate. Otherwise, natural selection will do.
Gene expression is inherently stochastic and pervasively regulated. While substantial work combining theory and experiments has been carried out to study how noise propagates through transcriptional ...regulations, the stochastic behavior of genes regulated at the level of translation is poorly understood. Here, we engineered a synthetic genetic system in which a target gene is down-regulated by a protein translation factor, which in turn is regulated transcriptionally. By monitoring both the expression of the regulator and the regulated gene at the single-cell level, we quantified the stochasticity of the system. We found that with a protein translation factor a tight repression can be achieved in single cells, noise propagation from gene to gene is buffered, and the regulated gene is sensitive in a nonlinear way to global perturbations in translation. A suitable mathematical model was instrumental to predict the transfer functions of the system. We also showed that a Gamma distribution parameterized with mesoscopic parameters, such as the mean expression and coefficient of variation, provides a deep analytical explanation about the system, displaying enough versatility to capture the cell-to-cell variability in genes regulated both transcriptionally and translationally. Overall, these results contribute to enlarge our understanding on stochastic gene expression, at the same time they provide design principles for synthetic biology.
A grand challenge in synthetic biology is to use our current knowledge of RNA science to perform the automatic engineering of completely synthetic sequences encoding functional RNAs in living cells. ...We report here a fully automated design methodology and experimental validation of synthetic RNA interaction circuits working in a cellular environment. The computational algorithm, based on a physicochemical model, produces novel RNA sequences by exploring the space of possible sequences compatible with predefined structures. We tested our methodology in Escherichia coli by designing several positive riboregulators with diverse structures and interaction models, suggesting that only the energy of formation and the activation energy (free energy barrier to overcome for initiating the hybridization reaction) are sufficient criteria to engineer RNA interaction and regulation in bacteria. The designed sequences exhibit nonsignificant similarity to any known noncoding RNA sequence. Our riboregulatory devices work independently and in combination with transcription regulation to create complex logic circuits. Our results demonstrate that a computational methodology based on first-principles can be used to engineer interacting RNAs with allosteric behavior in living cells.
The ability to engineer control systems of gene expression is instrumental for synthetic biology. Thus, bioinformatic methods that assist such engineering are appealing because they can guide the ...sequence design and prevent costly experimental screening. In particular, RNA is an ideal substrate to de novo design regulators of protein expression by following sequence-to-function models.
We have implemented a novel algorithm, RiboMaker, aimed at the computational, automated design of bacterial riboregulation. RiboMaker reads the sequence and structure specifications, which codify for a gene regulatory behaviour, and optimizes the sequences of a small regulatory RNA and a 5'-untranslated region for an efficient intermolecular interaction. To this end, it implements an evolutionary design strategy, where random mutations are selected according to a physicochemical model based on free energies. The resulting sequences can then be tested experimentally, providing a new tool for synthetic biology, and also for investigating the riboregulation principles in natural systems.
Web server is available at http://ribomaker.jaramillolab.org/. Source code, instructions and examples are freely available for download at http://sourceforge.net/projects/ribomaker/.
Synthetic biology aims at (re-)programming living cells like computers to perform new functions for a variety of applications. Initial work rested on transcription factors, but regulatory RNAs have ...recently gained much attention due to their high programmability. However, functional circuits mainly implemented with regulatory RNAs are quite limited. Here, we report the engineering of a fundamental arithmetic logic unit based on de novo riboregulation to sum two bits of information encoded in molecular concentrations. Our designer circuit robustly performs the intended computation in a living cell encoding the result as fluorescence amplitudes. The whole system exploits post-transcriptional control to switch on tightly silenced genes with small RNAs, together with allosteric transcription factors to sense the molecular signals. This important result demonstrates that regulatory RNAs can be key players in synthetic biology, and it paves the way for engineering more complex RNA-based biocomputers using this designer circuit as a building block.
MiRNAs have emerged as key regulators of stress response in plants, suggesting their potential as candidates for knock-in/out to improve stress tolerance in agricultural crops. Although diverse ...assays have been performed, systematic and detailed studies of miRNA expression and function during exposure to multiple environments in crops are limited.
Here, we present such pioneering analysis in melon plants in response to seven biotic and abiotic stress conditions. Deep-sequencing and computational approaches have identified twenty-four known miRNAs whose expression was significantly altered under at least one stress condition, observing that down-regulation was preponderant. Additionally, miRNA function was characterized by high scale degradome assays and quantitative RNA measurements over the intended target mRNAs, providing mechanistic insight. Clustering analysis provided evidence that eight miRNAs showed a broad response range under the stress conditions analyzed, whereas another eight miRNAs displayed a narrow response range. Transcription factors were predominantly targeted by stress-responsive miRNAs in melon. Furthermore, our results show that the miRNAs that are down-regulated upon stress predominantly have as targets genes that are known to participate in the stress response by the plant, whereas the miRNAs that are up-regulated control genes linked to development.
Altogether, this high-resolution analysis of miRNA-target interactions, combining experimental and computational work, Illustrates the close interplay between miRNAs and the response to diverse environmental conditions, in melon.
Cellular decision making is based on regulatory circuits that associate signal thresholds to specific physiological actions. This transmission of information is subjected to molecular noise what can ...decrease its fidelity. Here, we show instead how such intrinsic noise enhances information transfer in the presence of multiple circuit copies. The result is due to the contribution of noise to the generation of autonomous responses by each copy, which are altogether associated with a common decision. Moreover, factors that correlate the responses of the redundant units (extrinsic noise or regulatory cross-talk) contribute to reduce fidelity, while those that further uncouple them (heterogeneity within the copies) can lead to stronger information gain. Overall, our study emphasizes how the interplay of signal thresholding, redundancy, and noise influences the accuracy of cellular decision making. Understanding this interplay provides a basis to explain collective cell signaling mechanisms, and to engineer robust decisions with noisy genetic circuits.
Resumen La forma casi obsesiva en que el sistema capitalista tiende a asignar un valor de explotación a todo cuanto puede ser cuantificable, vinculada a las sucesivas evoluciones tecnológicas que han ...permitido la desmaterialización de los intercambios y la asignación de un valor a bienes intangibles, ha llegado al paroxismo de extrapolar dicha comprensión a los atributos de la personalidad. Este proceso de erosión de la esfera de derechos se da en beneficio de la capacidad monetizadora de un puñado de empresas que ostentan una posición monopólica transnacional. El artículo busca ofrecer respuestas a cuestiones legales que surgen respecto de las actividades de perfilamiento ( web tracking ) de ciudadanos en los contextos públicos y privados. A partir del examen crítico de las normas de protección al consumidor se plantean zonas de incorporación para nuevos conceptos que amplíen la protección de la privacidad.
Abstract The almost obsessive way in which the capitalist system tends to assign an exploitation value to everything that can be quantifiable and its linkage to the successive technological developments that have allowed the dematerialization of exchanges and the assignment of a value with respect to intangible goods has come to the paroxysm of extrapolating this understanding to the very attributes of personality. This process of erosion of the sphere of rights has occurred in favour of the monetizing capacity of a handful of companies that hold a transnational monopoly position. The article seeks to provide answers to legal questions that arise with respect to the web tracking activities of citizens in public and private contexts. Based on a critical examination of consumer protection regulations, areas of incorporation for new concepts that expand privacy protection are proposed.
Resumo A forma quase obsessiva como o sistema capitalista tende a atribuir um valor exploratório a tudo o que pode ser quantificado, associada às sucessivas evoluções tecnológicas que permitiram a desmaterialização das trocas e a atribuição de um valor a bens intangíveis, atingiu o paroxismo de extrapolar esse entendimento para os atributos de personalidade. Esse processo de erosão da esfera dos direitos beneficia a capacidade de monetização de empresas com uma posição de monopólio transnacional. Este artigo procura dar respostas a questões jurídicas que se levantam em relação às atividades de perfilamento dos cidadãos em contextos públicos e privados. Com base em um exame crítico dos regulamentos de proteção do consumidor, são propostas áreas de incorporação de novos conceitos que expandem a proteção da privacidade.
The population genetic mechanisms governing the preservation of gene duplicates, especially in the critical very initial phase, have remained largely unknown. Here, we demonstrate that gene ...duplication confers per se a weak selective advantage in scenarios of fitness trade-offs. Through a precise quantitative description of a model system, we show that a second gene copy serves to reduce gene expression inaccuracies derived from pervasive molecular noise and suboptimal gene regulation. We then reveal that such an accuracy in the phenotype yields a selective advantage in the order of 0.1% on average, which would allow the positive selection of gene duplication in populations with moderate/large sizes. This advantage is greater at higher noise levels and intermediate concentrations of the environmental molecule, when fitness trade-offs become more evident. Moreover, we discuss how the genome rearrangement rates greatly condition the eventual fixation of duplicates. Overall, our theoretical results highlight an original adaptive value for cells carrying new-born duplicates, broadly analyze the selective conditions that determine their early fates in different organisms, and reconcile population genetics with evolution by gene duplication.
The engineering of synthetic gene networks has mostly relied on the assembly of few characterized regulatory elements using rational design principles. It is of outmost importance to analyze the ...scalability and limits of such a design workflow. To analyze the design capabilities of libraries of regulatory elements, we have developed the first automated design approach that combines such elements to search the genotype space associated to a given phenotypic behavior. Herein, we calculated the designability of dynamical functions obtained from circuits assembled with a given genetic library. By designing circuits working as amplitude filters, pulse counters and oscillators, we could infer new mechanisms for such behaviors. We also highlighted the hierarchical design and the optimization of the interface between devices. We dissected the functional diversity of a constrained library and we found that even such libraries can provide a rich variety of behaviors. We also found that intrinsic noise slightly reduces the designability of digital circuits, but it increases the designability of oscillators. Finally, we analyzed the robust design as a strategy to counteract the evolvability and noise in gene expression of the engineered circuits within a cellular background, obtaining mechanisms for robustness through non-linear negative feedback loops.
PDF
