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.

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.

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.

Dynamic contrast-enhanced (DCE) time series analysis techniques are hard to fully validate quantitatively as ground truth microvascular parameters are difficult to obtain from patient data. This thesis presents a software application for generating synthetic image data from known ground truth tracer kinetic model parameters. As an object oriented design has been employed to maximise flexibility and extensibility, the application can be extended to include different vascular input functions, tracer kinetic models and imaging modalities. Data sets can be generated for different anatomical and motion descriptions as well as different ground truth parameters. The application has been used to generate a synthetic DCE-MRI time series of a liver tumour with non-linear motion of the abdominal organs due to breathing. The utility of the synthetic data has been demonstrated in several applications: in the development of an Akaike model selection technique for assessing the spatially varying characteristics of liver tumours; the robustness of model fitting and model selection to noise, partial volume effects and breathing motion in liver tumours; and the benefit of using model-driven registration to compensate for breathing motion. When applied to synthetic data with appropriate noise levels, the Akaike model selection technique can distinguish between the single-input extended Kety model for tumour and the dual-input Materne model for liver, and is robust to motion. A significant difference between median Akaike probability value in tumour and liver regions is also seen in 5/6 acquired data sets, with the extended Kety model selected for tumour. Knowledge of the ground truth distribution for the synthetic data was used to demonstrate that, whilst median Ktrans does not change significantly due to breathing motion, model-driven registration restored the structure of the Ktrans histogram and so could be beneficial to tumour heterogeneity assessments.

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.

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.

Remarkable progress has been made in our ability to design and produce synthetic DNA binding domains (TALE or Cas9-based), which can be further functionalized into synthetic transcription factors (sTFs). This technology is revolutionizing our ability to modulate expression of endogenous mammalian genes. Forced expression of cDNAs encoding transcription factors (TFs) is widely used to drive lineage conversions. However, this process is often inefficient and unreliable. Multiplex delivery of sTFs pool to activate endogenous master regulators and extinguish the expression profile of the host cell type could be a potential solution to this problem. We have developed a novel, simple TALE assembly method that enabled us to produce and screen large numbers of TAL effectors and compare their activity to dCas9-based TFs. During this process, we constructed many new functionally validated sTFs. Our ultimate goal is to test whether combining synthetic transcriptional activators and repressors can efficiently reprogram fibroblasts to NS cells or alternatively ‘program’ NS cell differentiation to neurons. We performed analyses of the transcriptome and chromatin accessibility of both fibroblasts and neural stem cells to unravel their core TF networks and their epigenetic state. This will allow us in the future the targeted design of sTFs and synthetic chromatin modifiers for specifically changing cell identity.

The reactions of carboxylic acids with organoaluminium reagents were studied, which led to the formation of novel aluminium compounds. The reactions of orthofunctionalised derivatives of benzoic acid with trivalent aluminium organyls AlR3, led to the formation of different Al-based molecular clusters, depending on the nature of R, the reaction stoichiometry and the character of the benzoic acid derivative. The obtained compounds were characterised in the solid state by X-ray diffraction methods and two main motifs were observed. When the acid and AlR3 reacted in a one-to-two stoichiometry the obtained products, [iBu4Al2(μ-O2CC6H4-2-μ- O)]2, [(Me2Al)2(μ-O2CC6H4-2-μ-NH)]2, [(iBu2Al)2(μ-O2CC6H4-2-μ-NH)]2, [(Me2Al)2(μ- O2CC6H4-2-μ-NMe)]2 and [(iBu2Al)2(μ-O2CC6H4-2-μ-NMe)]2, consisted of a central distorted 12-membered macrocycle, formed by two [Al-O-C-O-Al-X] units (X= O,N) and was found to be dimeric. The reaction between anthranilic acid derivatives and AlR3 could also take place in a one-to-one ratio. For anthranilic acid and Nmethylanthranilic acid the obtained crystals only allowed a qualitative analysis and showed the structure of the products, [MeAl(μ-O2CC6H4-2-μ-NH)]4, [iBuAl(μ-O2CC6H4- 2-μ-NMe)]4 to be tetrameric and each consisting of a distorted 16-membered ring formed by four [O-C-O-Al] units. With the reaction of N-phenylanthranilic acid it was possible to isolate a structural analogous product [iBuAl(μ-O2CC6H4-2-μ-NPh)]4 which could be fully characterised by x-ray crystallography and NMR spectroscopy. Where the quantity and quality of the obtained product was sufficient, the solution behaviour of the compounds was elucidated by multinuclear and multidimensional NMR spectroscopic techniques. The 27Al NMR showed that the aforementioned aggregates are maintained in solution, which for the 12-membered [Al-O-C-O-Al-N] macrocycle of [(iBu2Al)2(μ-O2CC6H4-2-μ-NH)]2 was confirmed by a NOESY spectrum. The second part of this project focused on the preliminary studies towards the application of aluminium compounds in the crosslinking of guar and carboxymethyl hydroxypropyl guar, which are common additives in hydraulic fracturing. Different commercially available aluminium compounds were tested for their general ability to crosslink the aforementioned polysaccharides, yielding promising results for aluminium lactate, aluminium acetylacetonate and aluminium isopropoxide. For the system comprising aluminium lactate in combination with CMHPG, rheological studies were carried out to determine the viscosity, the viscoelasticity, the shear recovery and the stability towards high temperatures. These sought to evaluate the crosslinking properties of the aluminium additive and to optimise the required conditions of the different system components. Finally, it was possible to obtain first proof-of-concept data suggesting that synthetically obtained aluminium compounds such as [Me2Al(μ- O2CPh)]2 and Al[MeC(CH2O)3]2(AlMe2)3 can be employed for the crosslinking of guar and CMHPG.

Interferometric Synthetic Aperture Radar (InSAR) is an established technique which has been applied to Earth surface displacement analysis and topographic reconstruction. Two complex coherent SAR acquisitions of the same scene are combined to form an interferogram from which surface displacement or terrain measurements are made. The similarities between both SAR signals is captured in the coherence and its magnitude is determined by the spatial separation between acquiring antennas and the changes (if any) to the physical characteristics of the scattering target in the duration between both SAR acquisitions. Both of these products derivable from the interferometric process have been applied in this study with the aim of enhancing monitoring and assessing changes in the coastal environment, with emphasis on the coastal geomorphology. A combination of remote sensing data acquired for Montrose Bay, NE Scotland, has been used to analyze changes to the geomorphology of the beach and dune system in terms of sediment volume analysis, erosion and accretion processes and shoreline changes over a short-term period of 4 years. The interferometric coherence was applied to detect changes to the dune morphology, which have been actively eroding at the southern flank of the Bay. The interferometric analysis presented in this thesis was based on SAR data acquired by the Sentinel-1 SAR antenna and the results demonstrated the limitations of the sensor for terrain mapping and DEM reconstruction. In addition, the significance of the vegetation on the interferometric coherence was demonstrated. However, the results have shown that temporal baseline remained a significant consideration in the application of interferometric coherence in highly dynamic environments such as the coastal environment.

We evaluate the external effects of 183 large mergers at the market level by assessing the impact on the main competitors of the merging firms. Using synthetic control groups and difference in difference estimation, we find that the return on assets of rival firms increases significantly after a merger. The size of the effect varies strongly with market characteristics and the intensity of competition.