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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

Combined Biosynthetic and Synthetic Production of Valuable Molecules: A Hybrid Approach to Vitamin E and Novel Ambroxan Derivatives

Adanve, Bertrand Tankpinou January 2015 (has links)
Synthetic chemistry has played a pivotal role in the evolution of modern life. More recently, the emerging field of synthetic biology holds the promise to bring about a paradigm shift with designer microbes to renewably synthesize complex molecules in a fraction of the time and cost. Still, given synthetic chemistry’s superior parsing powers to access a greater number of unnatural end products and nature’s virtuosity at stitching a staggering palette of carbon frameworks with ease, a hybrid approach that leverages the respective strengths of the two fields could prove advantageous for the efficient production of valuable natural molecules and their analogs. In a first demonstration of the hybrid approach where the biosynthesized intermediate is not part of the target molecule’s biosynthetic pathway, we engineered E. coli to produce Z,E-farnesol, which we subsequently transformed into a library of novel analogs of the commercially important amber fragrance Ambrox®, including the first synthetic patchouli scent. In a second demonstration of the hybrid approach, we produced the valuable tocotrienols (vitamin E) from yeast-produced geranylgeraniol in a single step C–C coupling with concomitant regioselective cycloetherification of the most proximal vinyl of the polyene, the first such process of its kind. The novel acid catalyst system that allowed for this unique regioselective cyclization holds promise as an asymmetric proton transfer tool and could open the door to facile asymmetric synthesis of vitamin E and other molecules.
42

Gene Regulatory Compatibility in Bacteria: Consequences for Synthetic Biology and Evolution

Johns, Nathan Isaac January 2019 (has links)
Mechanistic understanding of gene regulation is crucial for rational engineering of new genetic systems through synthetic biology. Genetic engineering efforts in new organisms are often hampered by a lack of knowledge about how regulatory components function in new host contexts. This dissertation focuses on efforts to overcome these challenges through the development of generalizable experimental methods for studying the behavior of DNA regulatory sequences in diverse species at large-scale. Chapter 2 describes experimental approaches for quantitatively assessing the functions of thousands of diverse natural regulatory sequences through a combination of metagenomic mining, high-throughput DNA synthesis and deep sequencing. By employing these methods in three distinct bacterial species, we revealed striking functional differences in gene regulatory capacity. We identified regulatory sequences with activity levels with activity levels spanning several orders of magnitude, which will aid in efforts to engineer diverse bacterial species. We also demonstrate functional species-selective gene circuits with programmable host behaviors that may be useful for microbial community engineering. In Chapter 3 we provide evidence for the evolution of altered stringency in σ70-mediated transcriptional activation based on patterns of initiation and activity from promoters of diverse compositions. We show that the contrast in GC content between a regulatory element and the host genome dictates both the likelihood and the magnitude of expression. We also discuss the potential implications of this proposed mechanism on horizontal gene transfer. The next two chapters focus on efforts aimed at extending the high-throughput methods described in earlier chapters to new organisms. Chapter 4 presents an in vitro approach for multiplexed gene expression profiling. Through the development and use of cell-free expression systems made from diverse bacteria, it was possible to rapidly acquire thousands of transcriptional measurements in small volume reactions, enabling functional comparisons of regulatory sequence function across multiple species. In Chapter 5 we characterize the restriction-modification system repertoires of several commensal bacterial species. We also describe ongoing efforts to develop methods for bypassing these systems in order to increase transformation efficiencies in species that are difficult or impossible to transform using current approaches.
43

Redirecting the cellular information flow with programmable dCas9-based chimeric receptors

Baeumler, Toni Andreas January 2018 (has links)
Signal integration and transduction by cell-surface receptors is a complex, multi-layered process resulting in tight regulation of downstream mediators, which in turn elicit pre-defined native cellular responses. The modular architecture of transmembrane receptors provides a unique opportunity for engineering de novo sensor/effector circuits, enabling the development of custom cellular functions for research and therapeutic applications. The signal transduction module of most existing chimeric receptors consists of either native intracellular domains or effectors domains fused to non-programmable DNA binding proteins. Therefore, these receptors can only engage in natural signalling pathways or drive the expression of artificial, pre-integrated transgenes. By harnessing the programmability of a nuclease deficient CRISPR/Cas9 (dCas9) signal transduction module and leveraging the evolutionarily optimised ligand-sensing capacity of native receptors, I have created a novel class of dCas9-based synthetic receptors (dCas9-synR). I demonstrate that an optimised split dCas9-based core architecture and custom protease-based signal release mechanism can be standardised across multiple classes of extracellular domains to engineer receptor tyrosine kinase (RTK)-based and G-protein-coupled receptor (GPCR)-based chimeric receptors. dCas9-synRTK and dCas9-synGPCR integrate a broad variety of input signals (peptides, proteins, lipids, sugars) with highly specific and robust activation of any custom output transcriptional programme in an agonist dose-dependent manner. Finally, to showcase the therapeutic potential of dCas9-synRs, I used them to convert a pro-angiogenic signal into an anti-angiogenic response, deploy a chemokine/cytokine programme in response to tumour-enriched biomolecules, and induce insulin expression following glucose stimulation. The performance of dCas9-synRs and their unique versatility in redirecting the information flow makes them ideally suited to engineer designer cells capable of sensing specific disease markers and in turn drive various therapeutic programmes.
44

Next generation approaches to polysaccharide preparation for Burkholderia pseudomallei vaccine development

Baldwin, Victoria Mae January 2016 (has links)
Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.
45

Synthetic biology in a fractiversal world : on novel biologies and modest geographies

Ledingham, Katie Anne January 2017 (has links)
The object of inquiry of this thesis is synthetic biology. In this thesis I ask what is this ‘thing’ that is synthetic biology (Latour, 2005) and what might it mean for synthetic biology to inhabit the world and to inhabit it well? Synthetic biology’s coming into being has been accompanied by a considerable amount of ‘hype’ and ‘hyperbole’ (Marris and Rose, 2012) – by what the philosopher Annemarie Mol (1999) would describe as a noisy ‘perspectivalism.’ My aim in this thesis is to contribute to the telling of different kinds of less-perspectival and less-technologically-deterministic stories about the development of this burgeoning approach to biological engineering. In drawing on a combination of empirical material from over 30 1-2 hour interviews with leading synthetic biologists and ethnographic materials generated from working alongside the UK’s Health and Safety Executive (the UK regulatory authority responsible for overseeing the development of synthetic biology), I aim to multiply outwards registers for understanding what synthetic biology is and what it might become. I highlight, for example, how synthetic biology is not simply a hubristic endeavour (Lewens, 2013) but is also about processes of learning and apprehension. What’s more, depending on how synthetic biology takes shape(s) in different practices, ‘time’ also becomes aleatory and freed from its modernist shackles (Serres, 2008). I use the lens of regulation as a means of addressing the question of what it might mean for synthetic biology to inhabit the world well. Synthetic biology’s regulatory provocations have been largely underexplored within STS and human geography literatures. The thesis is informed by and builds upon, theoretical notions of multiplicity (Mol, 2002) and of syncretisms (Law and Mol, 2013). The thesis contributes to a broader shift in social theory from critique towards compositionism and concludes by arguing for the development of a modest geography of novel biologies.
46

Mathematical model in absolute units for the Arabidopsis circadian oscillator

Urquiza García, José María Uriel January 2018 (has links)
The Earth’s oblique rotation results in changes in light and temperature across the day and time of year. Living organisms evolved rhythmic behaviours to anticipate these changes and execute appropriate responses at particular times. The current paradigm for the biological clocks in several branches of life is an underlying biochemical oscillator mainly composed by a network of repressive transcription factors. The slow decay in their activity is fundamental for generating anticipatory dynamics. Interestingly, these dynamics can be well appreciated when the biological system is left under constant environmental conditions, where oscillation of several physiological readouts persists with a period close to 24 hours, hence the term “circadian clocks”, circa=around dian=day. In plants the model species Arabidopsis thaliana has served as an invaluable tool for analysing the genetics, biochemical, developmental, and physiological effects of the oscillator. Many of these experimental results have been integrated in mechanistic and mathematical theories for the circadian oscillator. These models predict the timing of gene expression and protein presence in several genetic backgrounds and photoperiodic conditions. The aim of this work is the introduction of a correct mass scale for both the RNA transcript and protein variables of the clock models. The new mass scale is first introduced using published RNA data in absolute units, from qRT-PCR. This required reinterpreting several assumptions of an established clock model (P2011), resulting in an updated version named U2017. I evaluate the performance of the U2017 model in using data in absolute mass units, for the first time for this clock system. Introducing absolute units for the protein variables takes place by generating hypothetical protein data from the existing qRT-PCR data and comparing a data-driven model with western blot data from the literature. I explore the consequences of these predicted protein numbers for the model’s dynamics. The process required a meta-analysis of plant parameter values and genomic information, to interpret the biological relevance of the updated protein parameters. The predicted protein amounts justify, for example, the revised treatment of the Evening Complex in the U2017 model, compared to P2011. The difficulties of introducing absolute units for the protein components are discussed and components for experimental quantification are proposed. Validating the protein predictions required a new methodology for absolute quantification. The methodology is based on translational fusions with a luciferase reporter than has been little used in plants, NanoLUC. Firstly, the characterisation of NanoLUC as a new circadian reporter was explored using the clock gene BOA. The results show that this new system is a robust, sensitive and automatable approach for addressing quantitative biology questions. I selected five clock proteins CCA1, LHY, PRR7, TOC1 and LUX for absolute quantification using the new NanoLUC methodology. Functionality of translation fusions with NanoLUC was assessed by complementation experiments. The closest complementing line for each gene was selected to generate protein time series data. Absolute protein quantities were determined by generation of calibration curves using a recombinant NanoLUC standard. The developed methodology allows absolute quantification comparable to the calibrated qRT-PCR data. These experimental results test the predicted protein amounts and represent a technical resource to understand protein dynamics of Arabidopsis’ circadian oscillator quantitatively. The new experimental, meta-analysis and modelling results in absolute units allows future researchers to incorporate further, quantitative biochemical data.
47

Defining complex rule-based models in space and over time

Wilson-Kanamori, John Roger January 2015 (has links)
Computational biology seeks to understand complex spatio-temporal phenomena across multiple levels of structural and functional organisation. However, questions raised in this context are difficult to answer without modelling methodologies that are intuitive and approachable for non-expert users. Stochastic rule-based modelling languages such as Kappa have been the focus of recent attention in developing complex biological models that are nevertheless concise, comprehensible, and easily extensible. We look at further developing Kappa, in terms of how we might define complex models in both the spatial and the temporal axes. In defining complex models in space, we address the assumption that the reaction mixture of a Kappa model is homogeneous and well-mixed. We propose evolutions of the current iteration of Spatial Kappa to streamline the process of defining spatial structures for different modelling purposes. We also verify the existing implementation against established results in diffusion and narrow escape, thus laying the foundations for querying a wider range of spatial systems with greater confidence in the accuracy of the results. In defining complex models over time, we draw attention to how non-modelling specialists might define, verify, and analyse rules throughout a rigorous model development process. We propose structured visual methodologies for developing and maintaining knowledge base data structures, incorporating the information needed to construct a Kappa rule-based model. We further extend these methodologies to deal with biological systems defined by the activity of synthetic genetic parts, with the hope of providing tractable operations that allow multiple users to contribute to their development over time according to their area of expertise. Throughout the thesis we pursue the aim of bridging the divide between information sources such as literature and bioinformatics databases and the abstracting decisions inherent in a model. We consider methodologies for automating the construction of spatial models, providing traceable links from source to model element, and updating a model via an iterative and collaborative development process. By providing frameworks for modellers from multiple domains of expertise to work with the language, we reduce the entry barrier and open the field to further questions and new research.
48

Engineering and Delivery of Synthetic Chromatin Effectors

January 2019 (has links)
abstract: Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019
49

Engineering of polyketide biosynthetic pathways for bioactive molecules

Wang, Siyuan 01 May 2016 (has links)
Polyketides are a large group of structurally diverse natural products that have shown a variety of biological activities. These molecules are synthesized by polyketide synthases (PKSs). PKSs are classified into three types based on their sequence, primary structure, and catalytic mechanism. Because of the bioactivities of polyketide natural products, this study is focused on the engineering of PKS pathways for efficient production of useful bioactive molecules or structural modification to create new molecules for drug development. One goal of this research is to create an efficient method to produce pharmaceutically important molecules. Seven biosynthetic genes from plants and bacteria were used to establish a variety of complete biosynthetic pathways in Escherichia coli to make valuable plant natural products, including four phenylpropanoid acids, three bioactive natural stilbenoids, and three natural curcuminoids. A curcumin analog dicafferolmethane was synthesized by removing a methyltransferase from the curcumin biosynthetic pathway. Furthermore, introduction of a fungal flavin-dependent halogenase into the resveratrol biosynthetic pathway yielded a novel chlorinated molecule 2-chloro-resveratrol. This demonstrated that biosynthetic enzymes from different sources can be recombined like legos to make various plant natural products, which is more efficient (2-3 days) than traditional extraction from plants (months to years). Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in the first biosynthetic step of some plant phenylpropanoids. Based on the biosynthetic pathway of curcuminoids, a novel and efficient visible reporter assay was established for screening of phenylalanine ammonia-lyase (PAL) efficiency in Escherichia coli. The other goal of this research is to characterize and engineer natural product biosynthetic pathways for new bioactive molecules. The biosynthetic gene cluster of the antibacterial compound dutomycin was discovered from Streptomyces minoensis NRRL B-5482 through genome sequencing. Confirmation of the involvement of this gene cluster in dutomycin biosynthesis and creation of a series of new molecules were successfully conducted by rationally modifying the biosynthetic pathway. More importantly, a new demethylated analog of dutomycin was found to have much higher antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.
50

A Synthetic-biology Approach to Understanding Bacterial Programmed Death and Implications for Antibiotic Treatment

Tanouchi, Yu January 2013 (has links)
<p>Programmed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is `altruistic': the sacrifice of some cells can benefit the survivors through release of `public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, I determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. I further predicted that altruistic death could generate the `Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, I experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. These findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment.</p> / Dissertation

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