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.

Use of transcriptomic data for extending a model of the AppA/PpsR system in Rhodobacter sphaeroides.

Background: Photosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions.

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.

A single phosphatase can convert a robust step response into a graded, tunable or adaptive response.

Many biological signalling pathways have evolved to produce responses to environmental signals that are robust to fluctuations in protein copy number and noise. Whilst beneficial for biology, this robustness can be problematic for synthetic biologists wishing to re-engineer and subsequently tune the response of a given system. Here we show that the well-characterized EnvZ/OmpR two-component signalling system from Escherichia coli possesses one such robust step response.

Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides.

Chemotaxis is one of the best-characterized signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli.

Predicting inter-species cross-talk in two-component signalling systems.

Phosphosignalling pathways are an attractive option for the synthetic biologist looking for a wide repertoire of modular components from which to build. We demonstrate that two-component systems can be used in synthetic biology. However, their potential is limited by the fact that host cells contain many of their own phosphosignalling pathways and these may interact with, and cross-talk to, the introduced synthetic components.

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes.

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation.

Regulation of flagellum number by FliA and FlgM and role in biofilm formation by Rhodobacter sphaeroides.

The FlgM secretion checkpoint plays a crucial role in coordinating bacterial flagellar assembly. Here we identify a new role for FlgM and FliA as part of a complex regulatory network which controls flagellum number and is essential for efficient swimming and biofilm formation in the monotrichous bacterium Rhodobacter sphaeroides.

Genome sequence of Rhodobacter sphaeroides Strain WS8N.

Rhodobacter sphaeroides is a metabolically diverse photosynthetic alphaproteobacterium found ubiquitously in soil and freshwater habitats. Here we present the annotated genome sequence of R. sphaeroides WS8N.

New motion analysis system for characterization of the chemosensory response kinetics of Rhodobacter sphaeroides under different growth conditions.

We developed a new set of software tools that enable the speed and response kinetics of large numbers of tethered bacterial cells to be rapidly measured and analyzed. The software provides precision, accuracy, and a good signal-to-noise ratio combined with ease of data handling and processing. The software was tested on the single-cell chemosensory response kinetics of large numbers of Rhodobacter sphaeroides cells grown under either aerobic or photoheterotrophic conditions and either in chemostats or in batch cultures, allowing the effects of growth conditions on responses to be accurately measured.

Signal processing in complex chemotaxis pathways.

Bacteria use chemotaxis to migrate towards environments that are better for growth. Chemoreceptors detect changes in attractant levels and signal through two-component systems to control swimming direction. This basic pathway is conserved across all chemotactic bacteria and archaea; however, recent work combining systems biology and genome sequencing has started to elucidate the additional complexity of the process in many bacterial species.

Signal-dependent turnover of the bacterial flagellar switch protein FliM.

Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains approximately 13 different proteins and provides an ideal system to study functional molecular complexes.

Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes.

Fluorescence microscopy offers a minimally perturbative approach to probe biology in vivo. However, available techniques are limited both in sensitivity and temporal resolution for commonly used fluorescent proteins. Here we present a new imaging system with a diagnostic toolkit that caters for the detection and quantification of fluorescent proteins for use in fast functional imaging at the single-molecule level.

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment.

Rhodobacter sphaeroides: complexity in chemotactic signalling.

Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins.

Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli.

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na(+)-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na(+)).

In vivo and in vitro analysis of the Rhodobacter sphaeroides chemotaxis signaling complexes.

This chapter describes both the in vivo and in vitro methods that have been successfully used to analyze the chemotaxis pathways of R. sphaeroides, showing that two operons each encode a complete chemosensory pathway with each forming into independent signaling clusters. The methods used range from in vitro analysis of the chemotaxis phosphorylation reactions to protein localization experiments.

Stoichiometry and turnover in single, functioning membrane protein complexes.

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall.

The CheYs of Rhodobacter sphaeroides.

The Escherichia coli two-component chemosensory pathway has been extensively studied, and its response regulator, CheY, has become a paradigm for response regulators. However, unlike E. coli, most chemotactic nonenteric bacteria have multiple CheY homologues.

The positioning of cytoplasmic protein clusters in bacteria.

Cell division is a carefully orchestrated procedure. Bacterial cells have intricate mechanisms to ensure that genetic material is copied, proofread, and accurately partitioned into daughter cells. Partitioning now appears to also occur for some cytoplasmic proteins.

Requirements for chemotaxis protein localization in Rhodobacter sphaeroides.

Many proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium.

Localization of MreB in Rhodobacter sphaeroides under conditions causing changes in cell shape and membrane structure.

MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R.

Making sense of it all: bacterial chemotaxis.

Bacteria must be able to respond to a changing environment, and one way to respond is to move. The transduction of sensory signals alters the concentration of small phosphorylated response regulators that bind to the rotary flagellar motor and cause switching. This simple pathway has provided a paradigm for sensory systems in general.