Advancing synthetic biology to the multicellular level requires the development of multiple cell-to-cell communication channels that propagate information with minimal signal interference. The ...development of quorum-sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on cognate acyl-homoserine lactone (AHL)/transcription factor pairs, while the use of non-cognate pairs as a design feature has received limited attention. Here, we demonstrate a large library of AHL-receiver devices, with all cognate and non-cognate chemical signal interactions quantified, and we develop a software tool that automatically selects orthogonal communication channels. We use this approach to identify up to four orthogonal channels in silico, and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels is an enabling step that facilitates the design of synthetic consortia for applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation and spatial organisation.
Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that ...transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.
This paper employs dissipativity theory for the global analysis of limit cycles in particular dynamical systems of possibly high dimension. Oscillators are regarded as open systems that satisfy a ...particular dissipation inequality. It is shown that this characterization has implications for the global stability analysis of limit cycle oscillations: i) in isolated oscillators, ii) in interconnections of oscillators, and iii) for the global synchrony analysis in interconnections of identical oscillators
Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production ...for any given gene is a major challenge, as multiple processes and factors combine to determine translation efficiency. To enable prediction of the cost of gene expression in bacteria, we describe here a standard cell-free lysate assay that provides a relative measure of resource consumption when a protein coding sequence is expressed. These lysate measurements can then be used with a computational model of translation to predict the in vivo burden placed on growing E. coli cells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.
Heterologous gene expression can be a significant burden for cells. Here we describe an in vivo monitor that tracks changes in the capacity of Escherichia coli in real time and can be used to assay ...the burden imposed by synthetic constructs and their parts. We identify construct designs with reduced burden that predictably outperformed less efficient designs, despite having equivalent output.
The use of templates is a well-established method for the production of sequence-controlled assemblies, particularly long polymers. Templating is canonically envisioned as akin to a self-assembly ...process, wherein sequence-specific recognition interactions between a template and a pool of monomers favor the assembly of a particular polymer sequence at equilibrium. However, during the biogenesis of sequence-controlled polymers, template recognition interactions are transient; RNA and proteins detach spontaneously from their templates to perform their biological functions and allow template reuse. Breaking template recognition interactions puts the product sequence distribution far from equilibrium, since specific product formation can no longer rely on an equilibrium dominated by selective copy-template bonds. The rewards of engineering artificial polymer systems capable of spontaneously exhibiting nonequilibrium templating are large, but fields like DNA nanotechnology lack the requisite tools; the specificity and drive of conventional DNA reactions rely on product stability at equilibrium, sequestering any recognition interaction in products. The proposed alternative is handhold-mediated strand displacement (HMSD), a DNA-based reaction mechanism suited to producing out-of-equilibrium products. HMSD decouples the drive and specificity of the reaction by introducing a transient recognition interaction, the handhold. We measure the kinetics of 98 different HMSD systems to prove that handholds can accelerate displacement by 4 orders of magnitude without being sequestered in the final product. We then use HMSD to template the selective assembly of any one product DNA duplex from an ensemble of equally stable alternatives, generating a far-from-equilibrium output. HMSD thus brings DNA nanotechnology closer to the complexity of out-of-equilibrium biological systems.
This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on ...the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe an 'ECmax' assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with protein counting via fluorescence quenching, which we quantify and correct for the first time. Calibration across different instruments, disparate filter sets and mismatched gains is demonstrated to yield equivalent results. It also reveals that mCherry absorption at 600 nm does not confound cell density measurements unless expressed to over 100,000 proteins per cell. FPCountR is presented as pair of open access tools (protocol and R package) to enable the community to use this method, and ultimately to facilitate the quantitative characterisation of synthetic microbial circuits.
Predicting the evolution of engineered cell populations is a highly sought-after goal in biotechnology. While models of evolutionary dynamics are far from new, their application to synthetic systems ...is scarce where the vast combination of genetic parts and regulatory elements creates a unique challenge. To address this gap, we here-in present a framework that allows one to connect the DNA design of varied genetic devices with mutation spread in a growing cell population. Users can specify the functional parts of their system and the degree of mutation heterogeneity to explore, after which our model generates host-aware transition dynamics between different mutation phenotypes over time. We show how our framework can be used to generate insightful hypotheses across broad applications, from how a device's components can be tweaked to optimise long-term protein yield and genetic shelf life, to generating new design paradigms for gene regulatory networks that improve their functionality.
Highlights • Predictability and robustness are major challenges for synthetic biology. • Engineering bacteria requires putting synthetic constructs into growing host cells. • Synthetic constructs ...impact on the physiology and health of their host cells. • The physiology of the host cell impacts on the performance of synthetic constructs. • New tools to allow us to measure, reduce and predict host–construct interactions.
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