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Living organisms produce a wide range of metabolites. Because of their potential antibacterial, antifungal, antiviral, or cytostatic properties, such natural molecules are of high interest to the pharmaceutical industry. In nature, these metabolites are often synthesized via secondary metabolic biosynthetic gene clusters that are silent under the typical culturing conditions. Among different techniques used to activate these silent gene clusters, co-culturing of "producer" species with specific "inducer" microbes is a particularly appealing approach due to its simplicity. Although several "inducer-producer" microbial consortia have been reported in the literature and hundreds of different secondary metabolites with attractive biopharmaceutical properties have been described as a result of co-cultivating inducer-producer consortia, less attention has been devoted to the understanding of the mechanisms and possible means of induction for production of secondary metabolites in co-cultures. This lack of understanding of fundamental biological functions and inter-species interactions significantly limits the diversity and yield of valuable compounds using biological engineering tools. In this review, we summarize and categorize the known physiological mechanisms of production of secondary metabolites in inducer-producer consortia, and then discuss approaches that could be exploited to optimize the discovery and production of secondary metabolites.

Synthetic biology is a growing field at the interface of the engineering and biological sciences which aims to apply rational engineering design principles in biological contexts. It has many applications ranging from healthcare and the environment to industrial chemical synthesis and computation. When researchers attempt to apply synthetic biological tools to tackle these problems, they often follow a similar design-build-test cycle to that employed in traditional engineering disciplines. However, there are major challenges at each step in this cycle due to the variability inherent in biological systems, and our limited ability to understand and measure their behaviour. This thesis addresses select problems in each step of the design-build-test cycle as applied to synthetic biology, from a control-theoretic perspective. To aid the design process of synthetic biological circuits I begin by using traditional control-theoretic analysis to derive structural requirements that biological systems must satisfy if they are to achieve behaviours such as disturbance rejection. I demonstrate how these requirements can be used to inform the biological design process, and highlight the challenges that must be overcome for effective noise attenuation in such circuits. To refine the build/creation process for synthetic biological systems, I describe the experimental implementation and subsequent analysis of new synthetic biological feedback controllers built from two classes of components; short RNA and Recombinase proteins. Our results demonstrate how these circuits can be used to tune (or create) feedback loops, and reduce the influence of cellular noise on the performance of our synthetic circuits. This work thus has the double benefit of demonstrating new approaches to the engineering of biological systems, as well as creating well-characterised biological circuits that can be applied directly to a range of applications. Finally, to improve the testing/characterisation process for synthetic biological systems I develop a novel biological approach to measuring cell-to-cell variability, as well as a new automated experimental platform: The Turbidostat. This device provides many of the important measurement and actuation capabilities required for synthetic biological research in a cheap and easy-to-use robotic platform. It can thus be used to automate a diverse range of characterisation experiments that would otherwise be highly laborious and prone to variability/noise. I perform several validation experiments, demonstrating this device’s broad range of capabilities.

We evaluated immune biomarker profiles in HIV-infected adults (n=398) from 5 African countries. Although all biomarkers decreased after ART initiation, CXCL10, LBP, CRP, sCD163 and sCD14 were significantly higher during ART than in an HIV-negative reference group (n=90), indicating persistent monocyte/macrophage activation, inflammation and microbial translocation. Before ART initiation, high HIV viral load was associated with elevated CXCL10 and tuberculosis coinfection was associated with elevated sCD14. High pre-ART levels of each biomarker strongly predicted residual immune activation during ART. CCL2, LBP, CRP, IL-6 were differentially expressed between countries. Further research is needed on the clinical implications of residual immune dysregulation.

A simple aspirin-inducible system has been developed and characterised in E. coli by employing the Psal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that the effector free form SalRr could compete with the effector bound form SalRa, binding the Psal promoter and repress gene transcription. The induction of the Psal promoter was compared in two different gene-circuit designs: simple regulation system (SRS) and positive autoregulation (PAR). Both regulatory circuits were induced in a dose-dependent manner in the presence of aspirin in the range of 0.05-10 μM. Over-expression of SalR in the SRS circuit reduced both baseline leakiness and the strength of Psal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on SalRr/SalRa competitive binding model not only fit the observed experimental results in SRS and PAR circuits, but also predicted the performance of a new gene circuit design, for which weak expression of SalR in the SRS circuit should significantly improve the induction strength. The experimental result is in a good agreement with this prediction, validating the SalRr/SalRa competitive binding model. The aspirin-inducible systems were also functional in probiotic strain E.coli Nissle 1917 (EcN) and SimCells produced from E. coli MC1000 ΔminD. These well-characterised and modularised aspirin-inducible gene circuits would be useful biobricks to synthetic bology.

Major challenges exist in the design of gene regulatory networks. Some of these can be addressed by the in silico modelling and design of systems prior to implementation. However, reliable modelling of a given system is predicated upon a range of simplifying assumptions which may only be valid for a limited range of architectures and experimental conditions. In this paper we study the autorepressor, also referred to as the negative autoregulator, a genetic motif common both in natural and synthetic circuits. A number of approaches to modelling the autorepressor are presented, and one of these is extended to include the impact of inducer consumption, a phenomenon frequently observed in experiments. We implement this system using the tet-repressor (TetR), and compare the in vivo data with the results of simulations using parameters taken from the literature. We demonstrate that a modelling approach that considers inducer sequestration due its binding with a transcription factor may be required to qualitatively replicate experimental results. We conclude by drawing comparisons between experimental and simulated results, and discuss approaches by which modelling could be extended to better represent observed behaviours.

Accurate control of a biological process is essential for many critical functions in biology, from the cell cycle to proteome regulation. To achieve this, negative feedback is frequently employed to provide a highly robust and reliable output. Feedback is found throughout biology and technology, but due to challenges posed by its implementation, it is yet to be widely adopted in synthetic biology. In this paper we design a synthetic feedback network using a class of recombinase proteins called integrases, which can be re-engineered to flip the orientation of DNA segments in a digital manner. This system is highly orthogonal, and demonstrates a strong capability for regulating and reducing the expression variability of genes being transcribed under its control. An excisionase protein provides the negative feedback signal to close the loop in this system, by flipping DNA segments in the reverse direction. Our integrase/excisionase negative feedback system thus provides a modular architecture that can be tuned to suit applications throughout synthetic biology and biomanufacturing that require a highly robust and orthogonally controlled output.

Systemic immune activation has emerged as an essential component of the immunopathogenesis of HIV. It not only leads to faster disease progression, but also to accelerated decline of overall immune competence. HIV-associated immune activation is characterized by an increase in proinflammatory mediators, dysfunctional T regulatory cells, and a pattern of T-cell-senescent phenotypes similar to those seen in the elderly. These changes predispose HIV-infected persons to comorbid conditions that have been linked to immunosenescence and inflamm-ageing, such as atherosclerosis and cardiovascular disease, neurodegeneration, and cancer. In the antiretroviral treatment era, development of such non-AIDS-defining, age-related comorbidities is a major cause of morbidity and mortality. Treatment strategies aimed at curtailing persistent immune activation and inflammation may help prevent the development of these conditions. At present, the most effective strategy appears to be early antiretroviral treatment initiation. No other treatment interventions have been found effective in large-scale clinical trials, and no adjunctive treatment is currently recommended in international HIV treatment guidelines. This article reviews the role of systemic immune activation in the immunopathogenesis of HIV infection, its causes and the clinical implications linked to immunosenescence in adults, and the therapeutic interventions that have been investigated.