Regulation strategies for two-output biomolecular networks.

Feedback control theory facilitates the development of self-regulating systems with desired performance which are predictable and insensitive to disturbances. Feedback regulatory topologies are found in many natural systems and have been of key importance in the design of reliable synthetic bio-devices operating in complex biological environments. Here, we study control schemes for biomolecular processes with two outputs of interest, expanding previously described concepts based on single-output systems.

Deciphering mechanisms of production of natural compounds using inducer-producer microbial consortia.

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.

Dichotomous feedback: a signal sequestration-based feedback mechanism for biocontroller design.

We introduce a new design framework for implementing negative feedback regulation in synthetic biology, which we term 'dichotomous feedback'. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the process's architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realize than 'molecular sequestration' and other comparison motifs that are nowadays common in biomolecular feedback control design.

Reprogramming Synthetic Cells for Targeted Cancer Therapy.

Advances in synthetic biology enable the reprogramming of bacteria as smart agents to specifically target tumors and locally release anticancer drugs in a highly controlled manner. However, the bench-to-bedside translation of engineered bacteria is often impeded by genetic instability and the potential risk of uncontrollable replication of engineered bacteria inside the patient. SimCells (simple cells) are chromosome-free bacteria controlled by designed gene circuits, which can bypass the interference of the native gene network in bacteria and eliminate the risk of bacterial uncontrolled growth.

Genome-Scale Metabolic Modelling of Lifestyle Changes in Rhizobium leguminosarum.

Biological nitrogen fixation in rhizobium-legume symbioses is of major importance for sustainable agricultural practices. To establish a mutualistic relationship with their plant host, rhizobia transition from free-living bacteria in soil to growth down infection threads inside plant roots and finally differentiate into nitrogen-fixing bacteroids. We reconstructed a genome-scale metabolic model for Rhizobium leguminosarum and integrated the model with transcriptome, proteome, metabolome, and gene essentiality data to investigate nutrient uptake and metabolic fluxes characteristic of these different lifestyles.

Biomolecular mechanisms for signal differentiation.

Cells can sense temporal changes of molecular signals, allowing them to predict environmental variations and modulate their behavior. This paper elucidates biomolecular mechanisms of time derivative computation, facilitating the design of reliable synthetic differentiator devices for a variety of applications, ultimately expanding our understanding of cell behavior. In particular, we describe and analyze three alternative biomolecular topologies that are able to work as signal differentiators to input signals around their nominal operation.

Metabolic control of nitrogen fixation in rhizobium-legume symbioses.

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules.

Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis.

Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes.

In situ characterisation and manipulation of biological systems with Chi.Bio.

The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.

The effect of spatiotemporal antibiotic inhomogeneities on the evolution of resistance.

Combating the evolution of widespread antibiotic resistance is one of the most pressing challenges facing modern medicine. Recent research has demonstrated that the evolution of pathogens with high levels of resistance can be accelerated by spatial and temporal inhomogeneities in antibiotic concentration, which frequently arise in patients and the environment. Strategies to predict and counteract the effects of such inhomogeneities will be critical in the fight against resistance.

Low-Burden Biological Feedback Controllers for Near-Perfect Adaptation.

The robustness and reliability of synthetic biological systems can be substantially improved by the introduction of feedback control architectures that parallel those employed in traditional engineering disciplines. One common control goal is adaptation (or disturbance rejection), which refers to a system's ability to maintain a constant output despite variation in some of its constituent processes (as frequently occurs in noisy cellular environments) or external perturbations. In this paper, we propose and analyze a control architecture that employs integrase and excisionase proteins to invert regions of DNA and an mRNA-mRNA annihilation reaction.

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.

Development of Aspirin-Inducible Biosensors in and SimCells.

A simple aspirin-inducible system has been developed and characterized in by employing the promoter and SalR regulation system originally from ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in ADP1 suggests that the effector-free form, SalR, can compete with the effector-bound form, SalR, binding the promoter and repressing gene transcription. The induction of the promoter was compared in two different gene circuit designs: a simple regulation system (SRS) and positive autoregulation (PAR).

Synthetic negative feedback circuits using engineered small RNAs.

Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs).

Probing Intercell Variability Using Bulk Measurements.

The measurement of noise is critical when assessing the design and function of synthetic biological systems. Cell-to-cell variability can be quantified experimentally using single-cell measurement techniques such as flow cytometry and fluorescent microscopy. However, these approaches are costly and impractical for high-throughput parallelized experiments, which are frequently conducted using plate-reader devices.

A Dynamic Model of Resource Allocation in Response to the Presence of a Synthetic Construct.

Introducing synthetic constructs into bacteria often carries a burden that leads to reduced fitness and selective pressure for organisms to mutate their constructs and hence to a reduced functional lifetime. Understanding burden requires suitable methods for accurate measurement and quantification. We develop a dynamic growth model from physiologically relevant first-principles that allows parameters relevant to burden to be extracted from standard growth curves.

The Interplay between Feedback and Buffering in Cellular Homeostasis.

Buffering, the use of reservoirs of molecules to maintain concentrations of key molecular species, and negative feedback are the primary known mechanisms for robust homeostatic regulation. To our knowledge, however, the fundamental principles behind their combined effect have not been elucidated. Here, we study the interplay between buffering and negative feedback in the context of cellular homeostasis.

Ribo-attenuators: novel elements for reliable and modular riboswitch engineering.

Riboswitches are structural genetic regulatory elements that directly couple the sensing of small molecules to gene expression. They have considerable potential for applications throughout synthetic biology and bio-manufacturing as they are able to sense a wide range of small molecules and regulate gene expression in response. Despite over a decade of research they have yet to reach this considerable potential as they cannot yet be treated as modular components.

A Synthetic Recombinase-Based Feedback Loop Results in Robust Expression.

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.

Delineating parameter unidentifiabilities in complex models.

Scientists use mathematical modeling as a tool for understanding and predicting the properties of complex physical systems. In highly parametrized models there often exist relationships between parameters over which model predictions are identical, or nearly identical. These are known as structural or practical unidentifiabilities, respectively.

sbml-diff: A Tool for Visually Comparing SBML Models in Synthetic Biology.

We present sbml-diff, a tool that is able to read a model of a biochemical reaction network in SBML format and produce a range of diagrams showing different levels of detail. Each diagram type can be used to visualize a single model or to visually compare two or more models. The default view depicts species as ellipses, reactions as rectangles, rules as parallelograms, and events as diamonds.

Structural Identifiability of Dynamic Systems Biology Models.

A powerful way of gaining insight into biological systems is by creating a nonlinear differential equation model, which usually contains many unknown parameters. Such a model is called structurally identifiable if it is possible to determine the values of its parameters from measurements of the model outputs. Structural identifiability is a prerequisite for parameter estimation, and should be assessed before exploiting a model.

Designing Genetic Feedback Controllers.

By incorporating feedback around systems we wish to manipulate, it is possible to improve their performance and robustness properties to meet pre-specified design objectives. For decades control engineers have been successfully implementing feedback controllers for complex mechanical and electrical systems such as aircraft and sports cars. Natural biological systems use feedback extensively for regulation and adaptation but apart from the most basic designs, there is no systematic framework for designing feedback controllers in Synthetic Biology.

Designing Conservation Relations in Layered Synthetic Biomolecular Networks.

In Synthetic Biology, biomolecular networks are designed and constructed to perform specified tasks. Design strategies for these networks tend to center on tuning the parameters of mathematical models to achieve a specified behavior, and implementing these parameters experimentally. This design strategy often assumes a fixed network structure that defines the possible behaviors, which may be too restrictive for our purposes.

Simplified mechanistic models of gene regulation for analysis and design.

Simplified mechanistic models of gene regulation are fundamental to systems biology and essential for synthetic biology. However, conventional simplified models typically have outputs that are not directly measurable and are based on assumptions that do not often hold under experimental conditions. To resolve these issues, we propose a 'model reduction' methodology and simplified kinetic models of total mRNA and total protein concentration, which link measurements, models and biochemical mechanisms.