1,721,008 research outputs found
Overcoming Metabolic Burden in Synthetic Biology: a CRISPR interference approach
Full text linkSynthetic Biology is gaining an increasingly important role in the scientific community and dedicated research centers are rising all over the world. This discipline introduced the engineering principles of abstraction, modularity and standardization in the biology world; the application of these principles is allowing the design of complex biological systems to program living cells, realizing all sorts of desired function in many fields. These systems consist of DNA sequences, rationally combined to program the genetic instructions for cell behavior customization. Each part should behave as a biological brick for the design of complex genetic programs through functional building blocks; each module undergoes an extensive characterization to provide documentation on its functioning, enabling the rational design of complex circuits.
Mathematical modeling accompanies all the design procedure as a tool to describe the behavior of each single genetic module, in a bottom-up fashion that should allow the prediction of more complex systems obtained by the interconnection of pre-characterized parts. However, many unpredictability sources hamper the ideally rational design of those synthetic genetic devices, mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex biological living system.
Among others, the finite amount of translational resources in prokaryotic cells leads to an effect called metabolic burden, as a result of which hidden interactions between protein synthesis rates arise, leading to unexpected counterintuitive behaviors.
To face this issue, two actions have been proposed in this study: firstly, a recently proposed mathematical modeling solution that included a description of the metabolic load exerted by the expression of recombinant genes have been applied on a case study, highlighting its worth of use and working boundaries; second, a CRISPR interference-based architecture have been developed to be used as an alternative to high resource usage transcriptional protein regulators, studying the underlying mechanism in several circuital configurations and optimizing each forming part in order to achieve the desired specifications.
In Chapter 1, an introduction on synthetic biology is presented; in the second part, a brief overview on CRISPR technology and the overall aim of the study are reported.
In Chapter 2, a case study evaluating the use of mathematical modeling to properly include metabolic burden in rational design of a set of transcriptional regulator cascades is reported. Firstly, the circuits and expected behavior are introduced, along with the discussion about experimental data, dissenting from what initially predicted. Secondly, the comparison between the use of a classical Hill equation-based model and an improved version that explicitly consider the translational load exerted by the expression of recombinant genes is reported.
In Chapter 3, the design and deep characterization of a BioBrick-compatible CRISPR interference-based repression set of modules is shown; expression optimization of the molecular players is reported and its usability as a low-burden alternative is demonstrated with experimental data and mathematical modeling. Working boundaries, peculiar aspects and rooms for improvements are then highlighted.
In Chapter 4, preliminary studies aimed to improve the CRISPR interference system are reported and some of its context-dependencies are highlighted. Effects on repression efficiency due to alteration in the sequence of the RNA molecules addressing the CRISPR machinery to the desired target are discussed; evaluation of problems and opportunities related to the expression of more of this RNA guides are then highlighted. Lastly, an example of behavior of the system in presence of a competitor transcriptional regulator is reported.
In Chapter 5 the overall conclusions of this thesis work are drawn
Implementation of a Python simulator for microbial communities evolution via agent-based modeling
Full text linkRational engineering of protein- and CRISPRi- mediated regulation devices to design predictable interconnected circuits with reduced cell load
No full textQuorum Sensing Model Structures Inspire the Design of Quorum Quenching Strategies
No full textQuorum Sensing (QS) is a bacterial cell-to-cell communication mechanism allowing to share information about cell density, to adjust gene expression accordingly. Pathogens leverage QS to coordinate virulence and antimicrobial resistance, leading to distinctive population-level behaviors. To support rational design of synthetic biology strategies counteracting these mechanisms, we first mathematically model and compare two common QS architectures: one based on a single positive feedback loop to auto-induce signal molecule synthesis, the other including an additional positive feedback to increase signal molecule receptors production. Our comprehensive analysis of these QS structures and their equilibria highlights the differences in their bistable and hysteretic behaviors. An extensive sensitivity analysis is then performed, highlighting how parameter variations may lead to phenotype alterations in system behavior. Finally, building on our sensitivity analysis, we mathematically model four distinct QS inhibition strategies - signal molecule degradation, pharmaceutical inhibition, CRISPRi, and RNAi - which lead to the design of Quorum-Quenching (QQ) therapeutic approaches. Despite the underlying complex mechanisms, we demonstrate that the effect of the proposed QQ strategies can be captured by varying specific parameters within the QS models. We numerically analyze how these strategies affect the steady-state behavior of both QS models, identifying critical parameter thresholds for effective QS suppression
Simulating microbial communities' evolution via Agent base modelling: a Python tool
Full text linkIn this work, we blueprint a Dashboard that allows users to simulate the bacterial community's evolution through an intuitive GUI. The underlying Python-coded simulator implements an agent-based model of bacterial species, nutrients, and environment, allowing full customization and upgradability of the tool, due to its intrinsic modularity. Specifically, the model aims to represent discretized spaces, hosting a certain number of bacteria for each species and a defined amount of nutrients characterizing the surrounding environment. Bacteria can migrate from one spatial unit into another, looking for different nutrients (i.e., metabolites) across the whole space path. Growth and survival are governed by bacterial metabolisms, which are in turn functions of the metabolites present in each specific spatial unit at a certain time. Thus, our tool simulates how bacteria consume and produce metabolites, following species-specific metabolism rules, letting the system dynamically evolve through bacterial growth, death, spatial migration, and continuous updates of the available metabolite pool
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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