Tim Rudge

Computational modeling of synthetic microbial biofilms.

Microbial biofilms are complex, self-organized communities of bacteria, which employ physiological cooperation and spatial organization to increase both their metabolic efficiency and their resistance to changes in their local environment. These properties make biofilms an attractive target for engineering, particularly for the production of chemicals such as pharmaceutical ingredients or biofuels, with the potential to significantly improve yields and lower maintenance costs. Biofilms are also a major cause of persistent infection, and a better understanding of their organization could lead to new strategies for their disruption. Despite this potential, the design of synthetic biofilms remains a major challenge, due to the complex interplay between transcriptional regulation, intercellular signaling, and cell biophysics. Computational modeling could help to address this challenge by predicting the behavior of synthetic biofilms prior to their construction; however, multiscale modeling has so far not been achieved for realistic cell numbers. This paper presents a computational method for modeling synthetic microbial biofilms, which combines three-dimensional biophysical models of individual cells with models of genetic regulation and intercellular signaling. The method is implemented as a software tool (CellModeller), which uses parallel Graphics Processing Unit architectures to scale to more than 30,000 cells, typical of a 100 μm diameter colony, in 30 min of computation time.

A computational model of cellular morphogenesis in plants

Plant morphogenesis is the development of plant form and structure by coordinated cell division and growth. We present a dynamic computational model of plant morphogenesis at cellular level. The model is based on a self-reproducing cell, which has dynamic state parameters and spatial boundary geometry. Cell-cell signalling is simulated by diffusion of morphogens, and genetic regulation by a program or script. Each cell runs an identical script, equivalent to the genome. The model provides a platform to explore coupled interactions between genetic regulation, spatio-mechanical factors, and signal transduction in multicellular organisation. We demonstrate the capacity of the model to capture the key aspects of plant morphogenesis.

Fast image reconstruction in fluoresence optical tomography using data compression

We present a method for fast reconstruction in fluorescence optical tomography with very large data sets. In recent reports, CCD cameras at multiple positions have been used to collect optical measurements, producing more than 10(7) data samples. This makes storage of the full system Jacobian infeasible, and so data are usually subsampled. The method reported here allows use of the full data set, via image compression methods, and explicit construction of the (small) Jacobian, meaning optimal inversion methods can be applied, and thus leading to very fast reconstruction.

Full-wavelet approach for fluorescence diffuse optical tomography with structured illumination

We present a fast reconstruction method for fluorescence optical tomography with structured illumination. Our approach is based on the exploitation of the wavelet transform of the measurements acquired after wavelet-patterned illuminations. This method, validated on experimental data, enables us to significantly reduce the acquisition and computation times with respect to the classical scanning approach. Therefore, it could be particularly suited for in vivo applications.

Cell polarity-driven instability generates self-organized, fractal patterning of cell layers.

As a model system to study physical interactions in multicellular systems, we used layers of Escherichia coli cells, which exhibit little or no intrinsic coordination of growth. This system effectively isolates the effects of cell shape, growth, and division on spatial self-organization. Tracking the development of fluorescence-labeled cellular domains, we observed the emergence of striking fractal patterns with jagged, self-similar shapes. We then used a large-scale, cellular biophysical model to show that local instabilities due to polar cell-shape, repeatedly propagated by uniaxial growth and division, are responsible for generating this fractal geometry. Confirming this result, a mutant of E. coli with spherical shape forms smooth, nonfractal cellular domains. These results demonstrate that even populations of relatively simple bacterial cells can possess emergent properties due to purely physical interactions. Therefore, accurate physico-genetic models of cell growth will be essential for the design and understanding of genetically programmed multicellular systems.

Characterization of Intrinsic Properties of Promoters

Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.

Orthogonal intercellular signaling for programmed spatial behavior.

Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum‐sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell–cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3‐oxo‐C6‐ and 3‐oxo‐C12‐homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two‐channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long‐range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.

Wavelet-based data and solution compression for efficient image reconstruction in fluorescence diffuse optical tomography

Current fluorescence diffuse optical tomography (fDOT) systems can provide large data sets and, in addition, the unknown parameters to be estimated are so numerous that the sensitivity matrix is too large to store. Alternatively, iterative methods can be used, but they can be extremely slow at converging when dealing with large matrices. A few approaches suitable for the reconstruction of images from very large data sets have been developed. However, they either require explicit construction of the sensitivity matrix, suffer from slow computation times, or can only be applied to restricted geometries. We introduce a method for fast reconstruction in fDOT with large data and solution spaces, which preserves the resolution of the forward operator whilst compressing its representation. The method does not require construction of the full matrix, and thus allows storage and direct inversion of the explicitly constructed compressed system matrix. The method is tested using simulated and experimental data. Results show that the fDOT image reconstruction problem can be effectively compressed without significant loss of information and with the added advantage of reducing image noise.

Effects of Intrinsic and Extrinsic Noise Can Accelerate Juxtacrine Pattern Formation

Epithelial pattern formation is an important phenomenon that, for example, has roles in embryogenesis, development and wound-healing. The ligand Epithelial Growth Factor (EGF) and its receptor EGF-R, constitute a system that forms lateral induction patterns by juxtacrine signalling—binding of membrane-bound ligands to receptors on neighbouring cells. Owen et al. developed a generic ordinary differential equation model of juxtacrine lateral induction that exhibits stable patterning under some conditions. The model predicts relatively slow pattern formation. We examine here the effects of both intrinsic and extrinsic cellular noise arising from the stochastic treatment of this model, and show that this noise could have an accelerating effect on the patterning process.

Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies

Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.