A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities.

Engineered microbial consortia often have enhanced system performance and robustness compared with single-strain biomanufacturing production platforms. However, few tools are available for generating co-cultures of the model and key industrial host Saccharomyces cerevisiae. Here we engineer auxotrophic and overexpression yeast strains that can be used to create co-cultures through exchange of essential metabolites. Using these strains as modules, we engineered two- and three-member consortia using different cross-feeding architectures. Through a combination of ensemble modelling and experimentation, we explored how cellular (for example, metabolite production strength) and environmental (for example, initial population ratio, population density and extracellular supplementation) factors govern population dynamics in these systems. We tested the use of the toolkit in a division of labour biomanufacturing case study and show that it enables enhanced and tuneable antioxidant resveratrol production. We expect this toolkit to become a useful resource for a variety of applications in synthetic ecology and biomanufacturing.

Architectures for Combined Transcriptional and Translational Resource Allocation Controllers.

Recent work on engineering synthetic cellular circuitry has shown that non-regulatory interactions mediated by competition for gene expression resources can result in degraded performance or even failure. Transcriptional and translational resource allocation controllers based on orthogonal circuit-specific gene expression machinery have separately been shown to improve modularity and circuit performance. Here, we investigate the potential advantages, challenges, and design trade-offs involved in combining transcriptional and translational controllers into a "dual resource allocation control system." We show that separately functional, translational, and transcriptional controllers cannot generally be combined without extensive redesign. We analyze candidate architectures for direct design of dual resource allocation controllers and propose modifications to improve their performance (in terms of decoupling and expression level) and robustness. We show that dual controllers can be built that are composed only of orthogonal gene expression resources and demonstrate that such designs offer both superior performance and robustness characteristics.

Developing a graduate training program in Synthetic Biology: SynBioCDT.

This article presents the experience of a team of students and academics in developing a post-graduate training program in the new field of Synthetic Biology. Our Centre for Doctoral Training in Synthetic Biology (SynBioCDT) is an initiative funded by the United Kingdom's Research Councils of Engineering and Physical Sciences (EPSRC), and Biotechnology and Biological Sciences (BBSRC). SynBioCDT is a collaboration between the Universities of Oxford, Bristol and Warwick, and has been successfully running since 2014, training 78 students in this field. In this work, we discuss the organization of the taught, research and career development training. We also address the challenges faced when offering an interdisciplinary program. The article concludes with future directions to continue the development of the SynBioCDT.

Engineering Translational Resource Allocation Controllers: Mechanistic Models, Design Guidelines, and Potential Biological Implementations.

The use of orthogonal ribosomes in combination with dynamic resource allocation controllers is a promising approach for relieving the negative effects of cellular resource limitations on the modularity of synthetic gene circuits. Here, we develop a detailed mechanistic model of gene expression and resource allocation, which when simplified to a tractable level of complexity, allows the rational design of translational resource allocation controllers. Analysis of this model reveals a fundamental design trade-off: that reducing coupling acts to decrease gene expression. Through a sensitivity analysis of the experimentally tunable controller parameters, we identify how each controller design parameter affects the overall closed-loop behavior of the system, leading to a detailed set of design guidelines for optimally managing this trade-off. On the basis of our designs, we evaluated a number of alternative potential experimental implementations of the proposed system using commonly available biological components. Finally, we show that the controller is capable of dynamically allocating ribosomes as needed to restore modularity in a number of more complex synthetic circuits, such as the repressilator, and activation cascades composed of multiple interacting modules.

Dynamic allocation of orthogonal ribosomes facilitates uncoupling of co-expressed genes.

Introduction of synthetic circuits into microbes creates competition between circuit and host genes for shared cellular resources, such as ribosomes. This can lead to the emergence of unwanted coupling between the expression of different circuit genes, complicating the design process and potentially leading to circuit failure. By expressing a synthetic 16S rRNA with altered specificity, we can partition the ribosome pool into host-specific and circuit-specific activities. We show mathematically and experimentally that the effects of resource competition can be alleviated by targeting genes to different ribosomal pools. This division of labour can be used to increase flux through a metabolic pathway. We develop a model of cell physiology which is able to capture these observations and use it to design a dynamic resource allocation controller. When implemented, this controller acts to decouple genes by increasing orthogonal ribosome production as the demand for translational resources by a synthetic circuit increases.

Neural control of submucosal gland and apical membrane secretions in airways.

The mechanisms that lay behind the low-level secretions from airway submucosal glands and the surface epithelium in the absence of external innervation have been investigated in small areas (1.0-1.5 cm(2)) of mucosa from sheep tracheas, freshly collected from a local abattoir. Glandular secretion was measured by an optical method while short circuit current was used as a measure of surface secretion. Activation of neurones in the intrinsic nerve net by veratrine alkaloids caused an immediate increase in both glandular secretion and short circuit current, both effects being blocked by the addition of tetrodotoxin. However, agents known to be acting directly on the glands, such as muscarinic agonists (e.g., carbachol) or adenylate cyclase activators (e.g., forskolin) were not influenced by tetrodotoxin. The toxin alone had no discernable effect on the low-level basal secretion shown by unstimulated glands. Calu-3 cell monolayers, generally agreed to be a surrogate for the secretory cells of submucosal glands, showed no sensitivity to veratrine alkaloids, strengthening the view that the veratrine-like drugs acted exclusively on the intrinsic nerve net. The data are discussed in relation way in which transplanted lungs can maintain mucociliary clearance and hence a sterile environment in the absence of external innervation, as in transplanted lungs.