Synthetic biology practitioners look through an engineer's lens at the incredibly complex, sensitive and seemingly endless resource of living reproductive material and contemplate turning biology into a substrate – composed of modular, wellcharacterised parts – that can be used to design and build new functional devices and systems. It is often explained that this vision for engineering biology may deliver future forms of efficient drug production, renewable sources of biofuel, methods to sense and remediate toxins and numerous other applications. Yet, synthetic biology remains a field in its infancy, facing a barrage of interconnected challenges across technical, social, ethical, legal and political realms. This multifaceted dynamic makes it a timely and important locus for sociophilosophical investigation. This thesis provides a highly empirical ethnographic account of two research groups as they were challenged to design and build a microbiological machine for the International Genetically Engineered Machine competition (iGEM) in 2009. The work examines forms of knowledge and material production in synthetic biology and, in focusing on iGEM, argues that this field is not only a feat of technical engineering, but also one of social engineering as it educates and indoctrinates a next generation of researchers through this unique contest. In this narrative, one discovers a microsocial sphere in which new ideas and biological entities at the intersection of natural and synthetic kingdoms of life are being constructed. Forms of teaching, tools, practices and processes that make imagining, designing and building new living systems possible are illustrated. The reader is also introduced to some international stakeholders and dynamics at play. With gathering media interest, attention from art and design perspectives, as well as publications across social, philosophical, political and legal studies of this ‘new’ biotechnology, there is a great need for the kind of detailed, insider view that this thesis provides – it contributes to an informed space through which constructive questions may be asked as the debate around engineering synthetic life continues to unfold. As such, this work helps to enable a reflection on the kinds of intervention possible in the process of dreaming up ideas of potential future living machines. Involved collaborators, as well as the resistance of life itself, will ultimately govern the limits of synthetic biology.

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Laccase-catalysed depolymerisation of lignin offers substantial opportunities to form bio-derived aromatic chemicals. Laccase works with exquisite selectivity to oxidise the 5-5’ and β-5’ linkages in lignin and to cleave the β-O-4’ linkages in the presence of 1-hydroxybenzotriazole (1-HBT). However, the laccase/1-HBT system is inefficient when applied to industrial lignin, and the complexity of the product mixtures hinders rapid optimisation. Therefore, I studied laccase-catalysed oxidation of three synthetic lignin model oligomers, which had well-defined structures, yielded analytically tractable product mixtures, and, thus, permitted faster optimisation. Solvent systems were developed to dissolve the lignin model oligomers and retain laccase activity, which aided mass transfer. Models of “hardwood” (S:G 1:1) and “softwood” (G only) lignin containing nonphenolic β-O-4’ linkages (Angewandte Chemie-International Edition, 54, 258) were oxidised quickly by benzylic carbonyl formation ( > 60% in 24 h), or β-O-4’ bond cleavage. Approximately 25% of the linkages in the “hardwood” (S:G 1:1) oligomer were cleaved in 24 h. A more complex model of “softwood” lignin, containing β-5’, β-O-4’ and 5-5’ phenolic linkages (Green Chemistry, 15, 3031), was oxidised just as rapidly, but reaction products in this case were phenolic dimers that repolymerised. Therefore, I conclude that future bio-production of aromatic chemicals from lignin will depend on the development of improved lignin extraction procedures to allow retention of the β-O-4’ linkages, which will produce more efficient enzymatic depolymerisation.

The work presented in this thesis was initiated in order to develop a non-invasive real-time gas phase analytical technique, based on selected ion flow tube mass spectrometry (SIFT-MS), for monitoring the progression of human cell cultures and the detection of microbial contamination in such cultures by monitoring and quantifying the emitted volatile compounds. Fundamental SIFT experiments were performed to characterise the reactions of the SIFT-MS precursor ions (H3O+, NO+ and O2 +●) with several volatile compounds of potential value to biological research; a necessity for their quantification. The work has resulted in new methods for the quantification acetaldehyde and CO2 in gaseous samples. The compounds present in the headspace of sealed cultures of six human cell types were analysed by SIFT-MS, the key finding being their consumption of the toxic volatile compound acetaldehyde from the media. Further experiments involved the addition of the enzyme aldehyde dehydrogenase inhibitors diethylaminobenzaldehyde and disulfiram to cultures of hepG2 (hepatocellular carcinoma) cells, when it was observed that consumption of acetaldehyde from the cultures/headspace was reduced, and in some cases, acetaldehyde was even produced due to the actions of the cellular alcohol dehydrogenase enzyme. Furthermore, the solvent dimethylsulphoxide was reduced to imethylsulphide by the cells, which is known to occur via the enzyme methionine sulphoxide reductase. This process was retarded by the ALDH inhibitors. The use of SIFT-MS for the detection of microbial infection in mammalian cell cultures was also explored. The volatile compounds emitted by E. coli (strain JM109), into the gas phase above two different culture media, were analysed using SIFT-MS. Further, the progression of a culture of this bacterium was monitored continuously over a 4-hour period. The findings of this research were then applied to the study of human cell cultures intentionally infected by E. coli bacterium, including cultures contained in a 1L bioreactor.

The research carried out within this thesis was aimed at the development and implementation of a biocatalytic route towards Ticagrelor, a platelet-aggregation inhibitor. A bio-retrosynthetic consideration of the target compound yielded different possible strategies, which were analysed in terms of enantioselectivity and efficiency. The ultimate goal was to generate a biocatalyst specifically tailored to the starting material to yield the target compound in high optical purity and conversion. Different approaches to the chemoenzymatic generation of the cyclopropyl subunit (cf figure) in enantiomerically pure form were proposed and tested. The lipase from Thermomyces lanuginosus proved to be the most selective and active enzyme tested and was used as a model enzyme, initially yielding an E of 76 at a conversion of 50% after 48h. Through both reaction engineering and rational protein design approaches the time to attain 50% conversion could be reduced to 24 h while the enantioselectivity of the process increased to 100. Moreover, in a rational protein design approach different residues in the lid of the lipase were identified through analysis of the resolved crystal structures and subsequently mutated in order to investigate the influence of these residues on the overall performance of the lipase towards the target biotransformation. Mutations on Asn88 resulted in the inactivation of the enzyme while an Asp57Asn mutation resulted in a more active enzyme. Ultimately, this research has contributed to making the synthetic route towards Ticagrelor more environmentally sustainable, diminishing the need for the use of toxic, unsustainable and sterically demanding auxiliaries, as well as the amount of waste produced. The principles of green chemistry have been applied to the case studied. The synthetic route towards a key fragment of Ticagrelor has been significantly shortened using a biotransformation with an enzyme that can be recycled and employed in catalytic quantities.

In recent years biomolecules have been used to infer specific functionality to nanomaterials. Advances in conjugation techniques have allowed for the development of a vast range of hybrid bio-nano materials. Their applications range from biosensing and targeted therapy to metamaterials. In particular the conjugation of nanomaterials to functional oligonucleotides has been a thriving area of scientific research. In this project the main aim was to explore the uses of gold nanoparticle-DNA conjugates for biomedical applications. Probes for the real-time intracellular detection of mRNA were synthesized. These probes showed great target specificity, excellent biocompatibility and good cellular uptake. Importantly, unlike free nucleic acids, they displayed no susceptibility to degradation by nuclease enzymes. The ability to detect mRNAs in a live cell, in real time has tremendous diagnostic applications. Furthermore, multifunctional probes were designed. In addition to live cell mRNA detection, we developed probes with the ability to deliver a cytotoxic drug. Utilizing their inherent high specificity for target mRNAs, we demonstrated that cell-type specific targeted drug delivery was possible. In the absence of the target mRNA, the drug remained tightly bound within the probe. With a view of developing advanced materials, capable of performing multiple roles simultaneously, we investigated the use of nanoparticle assemblies for biomedical applications. In order to create highly stable nanoconstructs, a novel tool for the programmed assembly of DNA-nanomaterials was demonstrated. The use of copper-free click chemistry resulted in nano-assemblies connected by ssDNA. The employment of this novel tool proved to produce assemblies with covalently linked particles. Moreover, it was shown that gold nanoparticle dimers displayed excellent stability with respect to a variety of conditions commonly met within a biological environment. Additionally, the formation of heterogeneous nanoassemblies was demonstrated. Dimers of optical and either semi-conductor or magnetic nanocrystals were assembled representing examples of multi-role probes with exciting potential for applications in biomedicine.

Les nanoparticules fonctionnalisées continuent de susciter beaucoup d’interêt dans les applications biomédicales et les essais biologiques. Elles sont devenues un élément clé dans la recherche en nanobiotechnologie. Un des axes primordiaux des travaux de recherche est le développement de stratégies polyvalentes de fonctionnalisation de surface pour différentes nanoparticules allant de nanostructures de diamants à des nanoparticules d'oxyde de fer, des particules de silice et des nanocapsules lipidiques. Un des objectifs en particulier a été l’introduction de diverses fonctionnalisations sur les mêmes nanoparticules en utilisant soit des ligands dérivés de la dopamine ou soit par chimie « click » de Cu(I) catalysé. Il en résulte des nanostructures bien dispersées fonctionnalisées avec différents ligands à leurs surfaces. Les applications de ces nanostructures pour l'inhibition des infections virales et pour la délivrance de gènes ont été étudiées. En effet, l'inhibition de l'entrée du VHC a été identifiée comme étant une stratégie thérapeutique potentielle. Il a pu être démontré que différentes nanoparticules peuvent être efficacement conçues pour afficher les propriétés de lectine et se comporter donc comme des inhibiteurs efficaces d'entrée du virus in vitro. Les pseudo-lectines étudiées ici comprennent les nanoparticules dérivées du fer, de silice, du diamant et des nanocapsules lipidiques comportant toutes des fragments d’acide boronique attachés à leurs surfaces.Par ailleurs, le potentiel des nanoparticules de diamant pour la délivrance de gènes a été étudié. / Functionalized nanoparticles continue to attract interest in biomedical applications and bioassays and have become a key focus in nanobiotechnology research. One of the primal focuses of the research work was the development of versatile surface functionalization strategies for different nanoparticles ranging from diamond nanostructures to iron oxide nanoparticles, silica particles and lipid nanocapsules. One particular aim was the introduction of various functionalities onto the same nanoparticles using either dopamine-derived ligands or Cu(I) catalyzed “click” chemistry strategies. This resulted in well-dispersed nanostructures with different ligands present on the surface of the nanostructures. The possibilities to use such nanostructures for the inhibition of viral infections and for gene delivery were investigated. Indeed, inhibiting the entry of HCV has been identified as a potential therapeutic strategy. It could be demonstrated that various nanoparticles can be efficiently engineered to display “lectin-like” properties and indeed behave as effective viral entry inhibitors, in vitro. The pseudo-lectins investigated here include iron-, silica-, diamond-, (lipid nanocapsule)-derived nanoparticles all featuring surface-attached boronic acid moieties. In parallel to work on HCV entry inhibition, the potential of diamond nanoparticles as gene delivery system was investigated. Water dispersible and biocompatible polypegylated diamond particles were prepared using different dopamine ligands and their effect on gene delivery has been studied.

Les bactéries peuvent favoriser la formation de calcite. Leur utilisation au sein des matériaux cimentaires permettrait de colmater des fissures avec ce produit compatible et durable. L'objectif principal de cette thèse est de comprendre les mécanismes de la biocicatrisation des matériaux cimentaires afin d’accélérer la cinétique et maximiser le colmatage de fissures importantes (>200 µm). L'approche de biocicatrisation étudiée consiste à imprégner les fissures à l'aide d'un milieu de culture (milieu précurseur) contenant des bactéries. 3 aspects sont étudiés : les mécanismes abiotiques de cicatrisation (sans bactéries) des fissures de mortier, la croissance bactérienne in vitro, et la biocicatrisation des fissures de mortier. L'étude des mécanismes abiotiques porte sur l'autocicatrisation naturelle et sur l'influence des milieux précurseurs (urée, lactate de calcium et gluconate de calcium) sur la cicatrisation. L'influence d'une carbonatation initiale des fissures est aussi évaluée. L'étude sur les bactéries vise à suivre leur développement et à sélectionner le milieu de culture. L'étude de la biocicatrisation consiste à suivre la cicatrisation de fissures soumises aux traitements bactériens sélectionnés. Des fissures importantes (>200 µm) peuvent être réduites à 80 % par l'action des bactéries. Certains précurseurs ont un rôle important sur la cicatrisation de fissures fraichement ouvertes : en contact avec la pâte de ciment ils favorisent la formation d'ettringite qui comble partiellement les fissures. Il n'y a pas d'effet de ces précurseurs sur les fissures préalablement carbonatées. / Certain bacteria are able to promote calcite formation. Their use in the material allows the sealing of cracks by calcite, which is a compatible and durable product. The objective of this research is to achieve a better understanding of the biocicatrisation mechanisms in cementitious materials in order to accelerate its kinetics and maximize the sealing of large cracks (>200 µm). The studied bio-healing approach consists of soaking the cracks with a culture medium (precursor medium) containing bacteria. 3 aspects are investigated: abiotic healing mechanisms (without bacteria) of mortar cracks, in vitro bacterial growth, and bio-healing of mortar cracks. The study of abiotic mechanisms focuses on natural self-healing and addresses the influence of precursors (urea, calcium lactate, and calcium gluconate) on the healing process. The influence of the initial carbonation of cracks is also evaluated. The study of bacteria consists of monitoring bacterial growth and selecting the culture medium. The study of bio-healing aims to monitor the healing of cracks subjected to the selected bacterial treatments. Bacteria are improving the healing ability of the material. Large cracks (>200 µm) are reduced by 80 % through bacteria. Precipitation precursors have an important role in the healing of newly opened cracks: calcium lactate and calcium gluconate, in contact with the cement paste, promote the formation of ettringite, which partially fills the cracks. There are no effects of these precursors in previously carbonated cracks.

The seasonal variation of anammox is yet to be comprehensively studied, unlike denitrification, the more traditional sink for fixed nitrogen. A seasonal study of anammox, denitrification and benthic oxygen consumption using the revised isotope pairing technique is presented in Chapter 2. Experimental temperature and NO3- concentration were kept constant throughout so that the capacity of the sediment for anammox could be estimated. Similar seasonal variations in the rates of anammox, denitrification and oxygen consumption suggest that anammox is controlled by the availability of organic carbon. Furthermore the effect of tidal inundation by overlying water rich in NO3- was investigated by measuring rates of anammox, denitrification and oxygen consumption at three tidal elevations throughout the year. A significant relationship between anammox and denitrification was established at each tidal elevation, which increased in strength as length of inundation decreased. To complement this seasonal study, additional experiments were undertaken, which are described in Chapter 3, to determine how anammox, denitrification and sediment metabolism responds to variations in experimental NO3- concentration and temperature. There were significant increases in rates of anammox, denitrification and sediment metabolism with temperature until 20oC when rates of anammox began to reduce. Furthermore there was significant variation in the response of all three processes to temperature in samples collected at different dates, which suggested that reduced bioavailability of organic carbon in the winter months was limiting the response to temperature. In addition to exploring how inorganic N is cycled in estuarine sediments, the ability of estuarine sediments to oxidize urea via nitrite was examined using 15N and 13C labelled substrates. Results, which are presented in Chapter 4, indicate that urea added to anaerobic sediment slurries was rapidly hydrolysed to ammonium before being oxidized via the anammox pathway.

Toxicity of organic solvents to microbial hosts is a major consideration in the economical production of biofuels such as ethanol and especially butanol, with low product concentrations leading to high recovery costs. The key to rational engineering of solvent tolerant microorganisms for such processes lies in obtaining appropriate tolerance genes (modules) suited for different compounds. In this project, a synthetic biology approach was adopted to generate a library of standardised BioBrick parts involved in different stress responses. Using a multiple-assay approach, including a bioluminescence assay, these stress response genes were tested individually and in combination to determine their effects on survival in ethanol, nbutanol, acetone and fermentation inhibitors produced by biomass pre-treatment. A set of tolerance modules was obtained for ethanol and n-butanol. Proof-of-concept tests suggested that ethanol and n-butanol toxicity was mainly due to damage to membrane, cellular proteins and DNA possibly by oxidative stress. No synergistic interactions were observed from a combination of different tolerance genes. Further tests carried out using enzyme and fluorescence-based assays to elucidate the effect of n-butanol on the cell envelope showed that the solvent released lipopolysaccharides from the outer membrane of E. coli and also caused both outer and inner membranes to be leaky. Very high n-butanol concentrations resulted in an altered cell shape and bleb formation suggesting an impairment in cell division and peptidoglycan biosynthesis. The cell membrane was modified by cis-trans isomerisation of unsaturated fatty acids in the phospholipids resulting in a reduction of membrane leakage which in effect, increased n-butanol tolerance in E. coli. In conclusion, results from this research suggest that strategies to protect the membrane and cellular proteins should be included in rational engineering of n-butanol tolerant bacteria.