For the field of synthetic biology, the adaptation of principles, from the well established traditional engineering disciplines, like mechanical and electrical engineering, in order to realise complex synthetic biological circuits, is an intriguing prospect. These principles can enable a forward engineering, rational design and implementation approach, where a system's properties can be predicted or designed in silico followed by the manufacturing of the in vivo system, that can be tested, used or redesigned in the most efficient possible way. Achieving control over these circuits, is one of the important topics of the field, for these applications to become robust and render useful functions applicable to energy, medicine, pharmaceuticals and agriculture industries. In this work, I attempt to explore light, as a promising control 'dial' for synthetic circuitry. Light is fast, economic compared to chemicals, it can be interfaced with electronics, it is reversible in its effect and can be applied at a fine spatio-temporal resolution. These characteristics, are absent from the classically used chemical inducers, meaning that light, can open new possibilities for the user to control synthetic systems, or even facilitate the cell to cell communication, within population based networks. This work, is a contribution towards harnessing the advantages of light, for achieving control over synthetic circuits. More specifically, I start with the detailed theoretical and experimental study of the Cph8 two component system, a synthetic chimeric receptor which is responsive to red light. This is done, in order to develop a sufficient theoretical understanding of it, through detailed mechanistic modelling, in order to connect the specific system with the toggle switch and the dual feedback oscillator, in an optimal way and achieve control of these devices through light. The developed model, was able to highlight the main aspects and mechanisms inherent to its structure, describe most of the observations from the experimental system, to also make quantitative predictions. The second part of this work, was the development of novel promoters, that can be regulated by a commonly used transcription factor, such as LacI, but also, light responsive regulators like OmpR and CcaR. This yielded a direct way to integrate light and chemical inputs, into a single output, while the dual regulation, allowed to connect and modulate the toggle switch without the need of additional transcription factors. The latter, a light tuneable toggle switch, showed indications that it can function as a memory controller that can be reset by light. Finally, I show the design and modelling of a light tuneable dual feedback oscillator, where light of one wavelength can be used to tune the amplitude, while another wavelength can tune the period. The developed models and synthetic circuits are expected to contribute towards implementing finely tuned and controlled synthetic circuits through light.

There has been much recent interest in using ionic liquids for processing lignocellulosic biomass. While cellulose has an acknowledged application in generating biofuels, it would be valuable to use the abundant lignin present as well. Rhodococcus has been reported previously to degrade lignin. Therefore, it is attractive to consider a scheme in which an ionic liquid is also used to enhance the microbial breakdown of lignin (bioligninolysis). By using vanillic acid as model compound (Chapter 3), results showed that Rhodococcus UKMP-5M is able to degrade vanillic acid as a sole carbon source at 10mM concentration to give the highest growth rate. An oxygen-dependent reaction degrades vanillic acid into protocatechuic acid and formate in a previously undescribed metabolic pathway. In Chapter 4, GC-MS demonstrated guaiacol as the major product of lignin degradation and the lignin degradation assay indicates that the treatment of lignin with ionic liquids assist the lignin degradation despite some ionic liquids showing a toxicity effect on the cells. Toxicological studies (Chapter 5) demonstrated different ionic liquids show varying toxicity to the bacteria. By using classical disk diffusion test in screening 16 different ionic liquids, it was revealed that the toxicity is correlated with the size of the ionic alkyl chain; however, the carbon atom count, not the structure or the distribution of those atoms in the cation, correlates directly with the toxicity. The strongest link was discovered with pH effects rather than with structure in the toxicity of acidic ionic liquids. We also propose that the octanol-water partition coefficient (Kow) has the controlling impact on the toxicity of ionic liquids. Bacterial growth curves exhibited three different trends: a complete inhibition, an increase in toxicity with an increase of ionic liquid concentration and the extension of the lag phase due to bacterial adaptation. This study established that Rhodococcus UKMP-5M could adapt and grow in the presence of ionic liquids with 1-ethyl-3-methylimidazolium acetate, [Emim][OAc] as the most promising candidate in designing an ionic liquid-facilitated system of bioligninolysis.

Synthetic Biology represents a movement to utilise biological organisms for novel applications through the use of rigorous engineering principles. These principles rely on a solid and well versed understanding of the underlying biological components and functions (relevant to the application). In order to achieve this understanding, reliable behavioural and contextual information is required (more commonly known as characterisation data). Focussing on lowering the barrier of entry for current research facilities to regularly and easily perform characterisation assays will directly improve the communal knowledge base for Synthetic Biology and enable the further application of rational engineering principles. Whilst characterisation remains a fundamental principle for Synthetic Biology research, the high time costs, subjective measurement protocols, and ambiguous data analysis specifications, deter regular performance of characterisation assays. Vitally, this prevents the valid application of many of the key Synthetic Biology processes that have been derived to improve research yield (with regards to solving application problems) and directly prevent the intended goal of addressing the ad hoc nature of modern research from being realised. Designing new technologies and tools to facilitate rapid 'hands off' characterisation assays for research facilities will improve the uptake of characterisation within the research pipeline. To achieve this two core problem areas have been identified that limit current characterisation attempts in conventional research. Therefore, it was the primary aim of this investigation to overcome these two core problems to promote regular characterisation. The first issue identified as preventing the regular use of characterisation assays was the user-intensive methodologies and technologies available to researchers. There is currently no standardised characterisation equipment for assaying samples and the methodologies are heavily dependent on the researcher and their application for successful and complete characterisation. This study proposed a novel high throughput solution to the characterisation problem that was capable of low cost, concurrent, and rapid characterisation of simple biological DNA elements. By combining in vitro transcription-translation with microfluidics a potent solution to the characterisation problem was proposed. By utilising a completely in vitro approach along with excellent control abilities of microfluidic technologies, a prototype platform for high throughput characterisation was developed. The second issue identified was the lack of flexible, versatile software designed specifically for the data handling needs that are quickly arising within the characterisation speciality. The lack of general solutions in this area is problematic because of the increasing amount of data that is both required and generated for the characterisation output to be considered as rigorous and of value. To alleviate this issue a novel framework for laboratory data handling was developed that employs a plugin strategy for data submission and analysis. Employing a plugin strategy improves the shelf life of data handling software by allowing it to grow with the needs of the speciality. Another advantage to this strategy is the increased ability for well documented processing and analysis standards to arise that are available for all researchers. Finally, the software provided a powerful and flexible data storage schema that allowed all currently conceivable characterisation data types to be stored in a well-documented manner. The two solutions identified within this study increase the amount of enabling tools and technologies available to researchers within Synthetic Biology, which in turn will increase the uptake of regular characterisation. Consequently, this will potentially improve the lateral transfer of knowledge between research projects and reduce the need to perform ad hoc experiments to investigate facets of the fundamental biological components being utilised.

Solvate formation is a phenomenon that has received special attention in solid state chemistry over the past few years. This is due to its potential to both improve and impair pharmaceutical formulations. The reasons for solvate formation aren’t explicitly known. Therefore, there is currently no reliable guide in the literature on what solvents to choose in order to avoid or form a solvate when crystallizing an organic material. In this thesis we address the problem by trying to find the main reasons of solvate formation. A knowledge-based approach was used to link the molecular structure of an organic compound to its ability to form a solvate with five different solvents; these are ethanol, methanol, dichloromethane, chloroform and water. The Cambridge Structural Database (CSD) was used as a source of information for this study. A supervised machine learning method, logistic regression was found to be the optimal method for fitting these knowledge-based models. The result was one predictive model per solvent, with a success rate of 74-80 %. Each model incorporated two molecular descriptors, representing two molecular features of molecules. These are the size and branching in addition to hydrogen bonding ability. The models’ predictive ability was validated via experimental work, in which slurries of 10 pharmaceutically active ingredients were screened for solvate formation with each of the five solvents in the study. During the screening process, a new diflunisal dichloromethane solvate, a diflunisal chloroform solvate and a hymercromone methanol solvate were found. The PXRD patterns of these forms are reported. The thesis also includes SCXRD analysis of a previously known grisoefulvin dichloromethane solvate, a previously known fenofibrate polymorph and a new fenofibrate polymorph.

No description available.

Synthetic biology, an engineering approach to genetic modification, has emerged at a time when academics are increasingly expected to translate research to other domains of society. Proponents of synthetic biology often deploy promissory rhetoric to create expectations of major improvements in medicine, energy and food production. How else are actors in the field of synthetic biology addressing these translational expectations? This thesis takes synthetic biology in the UK as an empirical site to explore the various ways in which research translation involves multiple rhetorical, organisational and material transformations. In this project I developed a conceptual framework using post-Actor Network Theory, post-social theory and other STS concepts. I generated data by employing qualitative research methods including observations, interviews and by collecting documentation from various institutions. I visited field sites such as academic science laboratories, academic events and administrative offices. Participants included scientific researchers, research administrators, industry representatives and policymakers. I transcribed the interview data, typed up field notes and iteratively coded the texts and documents to generate themes. From my analysis I identified a variety of strategies and practices that appear to make synthetic biology translatable. These included: articulating synthetic biology research with absences in other areas of society (e.g. state economic and industrial deficits, problems with private-public collaborations) and imagining a future industry; demarcating synthetic biology research from other programmes such as genetically modified organisms; realising rhetorical promises in the everyday organisation, research training and material work of synthetic biology practices. My research indicates that translation in synthetic biology involves multiple groups orientating research facilities and researcher training, particularly towards industrial manufacturing. I go on to theorise synthetic biology as an unfolding multiple. Actors expand synthetic biology and in the process they entangle the state, institutions, laboratories, cells and molecules. To achieve this, actors mobilise vulnerabilities that others have identified in science, state and society to create a central heroic object of synthetic biology. These conclusions offer a conceptual framework to further investigate and interpret contemporary technoscience and its connections in society.

No description available.

The work reported in this thesis was part of the larger EPSRC (Engineering and Physical Sciences Research Council) and dstl (Defence Science and Technology Laboratory) funded project called CRASHCOMPS with the main aim to establish approaches for crack arrest and self healing in composites. The aim of this subproject was to produce macroporous polymer beads with either open pore or closed-cell structures, which can be utilised as delivery vehicles for crush-release of self-healing agents but which could ultimately also be used for crush-release of other active ingredients, such as dyes, drugs, enzyme, acids etc. The work reported in this thesis describes the synthesis and characterisation of novel micrometre sized macroporous polymer beads which can be easily handled and dispersed in liquids, gels, epoxy resins and particle blends and are therefore potentially useful for a large variety of applications. Throughout this thesis, macroporous poly(methyl methacrylate-co-ethylene glycol dimethacrylate) beads were synthesised via double emulsion templating in combination with suspension and microfluidic polymerisation techniques as it allowed the synthesis of macroporous polymer beads with predefined pore structures and bead sizes, respectively. By using different internal phase volume ratios and surfactants of different concentration for stabilisation of the primary emulsions, macroporous polymer beads with different open pore morphologies and mechanical properties could be synthesised. Furthermore by using particles as emulsifiers for the primary emulsion templates, macroporous beads with closed-cell structures and significant improvement in the mechanical properties of resulting beads could be realised, which are important for crush-release applications. This work led to the formation of novel high internal phase emulsions (HIPEs), which use active ingredients, such as hydrochloric acid or guar gum degrading enzymes, as internal emulsion phase and which allowed to produce in situ filled macroporous polymer beads. These beads then allowed the release of the active ingredients upon crushing. Subsequently, the ambitious aim to use macroporous beads as carriers for self-healing agents was approached and synthesised macroporous beads were post filled with epoxy resin and sealed afterwards. In addition, macroporous beads filled with epoxy resin were incorporated into composite matrices and their effect on the overall mechanical properties as well as the amount of unreacted resin available for self-healing approaches after curing of the composite investigated and determined, respectively. Eventually novel hydrophilic macroporous polymer monoliths based on [2-(acryloyloxy)ethyl] trimethylammonium chloride and 2-(methacryloyloxy)ethyl trimethylammonium chloride with N,N?-methylenebis(acrylamide) as crosslinker were produced and characterised for potential in situ encapsulation with epoxy resin.

Saturation mutagenesis is a powerful tool in modern protein engineering, which permits key residues within a protein to be targeted in order to potentially enhance specific functionalities. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK/S) has inherent redundancy and consequent disparities in codon representation. Therefore, both chemical (trinucleotide phosphoramidites) and biological methods (sequential, enzymatic single codon additions) of non-degenerate saturation mutagenesis have been developed in order to combat these issues and so improve library quality. Large libraries with multiple saturated positions can be limited by the method used to screen them. Although the traditional screening method of choice, cell-dependent methods, such as phage display, are limited by the need for transformation. A number of cell-free screening methods, such as CIS display, which link the screened phenotype with the encoded genotype, have the capability of screening libraries with up to 1014 members. This thesis describes the further development of ProxiMAX technology to reduce library codon bias and its integration with CIS display to screen the resulting library. Synthetic MAX oligonucleotides are ligated to an acceptor base sequence, amplified, and digested, subsequently adding a randomised codon to the acceptor, which forms an iterative cycle using the digested product of the previous cycle as the base sequence for the next. Initial use of ProxiMAX highlighted areas of the process where changes could be implemented in order to improve the codon representation in the final library. The refined process was used to construct a monomeric anti-NGF peptide library, based on two proprietary dimeric peptides (Isogenica) that bind NGF. The resulting library showed greatly improved codon representation that equated to a theoretical diversity of ~69%. The library was subsequently screened using CIS display and the discovered peptides assessed for NGF-TrkA inhibition by ELISA. Despite binding to TrkA, these peptides showed lower levels of inhibition of the NGF-TrkA interaction than the parental dimeric peptides, highlighting the importance of dimerization for inhibition of NGF-TrkA binding.

The aim of this thesis was to improve the formulation processes of an existing oral vaccine delivery system, the bilosome, and to investigate its mechanism of action. There were three main areas of research; (1) refinement and adaptation of the existing homogenisation melt method, (2) development of a new formulation process, and (3) investigation of the mechanism of action. The results from (1) showed that lyophilisation has no detrimental effect on the bilosome, allowing improved storage characteristics; this was proven in a 9-month study which showed that the immunogenicity of the lyophilised formulation was retained after this time. With a view to developing a system which could be more easily mass produced,a new formulation process using a microwave reactor was developed in (2). This gave bilosomes with equal immunogenicity to those in (1), in fewer steps and 1/5th of the time; these also allowed incorporation of inexpensive surfactants, which was not possible with the original method. As the formulation process had been successfully streamlined, the mechanism of action was examined in (3). It was thought that further understanding of this could provide information which would allow enhancement of the bilosomes immunogenicity. Results showed that no enhancement of immunogenicity was possible using bilosomes incorporating squalene, or with suppression of gastric acid pre-administration. Investigation of uptake in the intestine showed uptake in both the villi and the Peyer’s patches of the small intestine,which may prove useful in the development of future vaccine delivery systems. The study in the lungs was less successful, and a number of issues meant that no significant conclusions could be made; however, the groundwork has been laid for future work in this area.