Imperial College London

Guy-Bart Stan

Faculty of EngineeringDepartment of Bioengineering

Professor of BioSystems Engineering & Control
 
 
 
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Contact

 

+44 (0)20 7594 6375g.stan Website

 
 
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Location

 

B703Royal School of MinesSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

142 results found

Bubnov DM, Yuzbashev TV, Khozov AA, Melkina OE, Vybornaya TV, Stan G-B, Sineoky SPet al., 2022, Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs., Nucleic Acids Research, Vol: 50, ISSN: 0305-1048

Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50-100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

Journal article

Ledesma Amaro R, Stan G-B, Atkinson E, Perrino G, Tuza Zet al., 2022, Resource-aware whole-cell model of division of labour in a two-strain consortium for complex substrate degradation, Microbial Cell Factories, Vol: 21, Pages: 1-12, ISSN: 1475-2859

BackgroundLow-cost sustainable feedstocks are essential for commercially viable biotechnologies. These feedstocks, often derived from plant or food waste, contain a multitude of different complex biomolecules which require multiple enzymes to hydrolyse and metabolise. Current standard biotechnology uses monocultures in which a single host expresses all the proteins required for the consolidated bioprocess. However, these hosts have limited capacity for expressing proteins before growth is impacted. This limitation may be overcome by utilising division of labour (DOL) in a consortium, where each member expresses a single protein of a longer degradation pathway.ResultsHere, we model a two-strain consortium, with one strain expressing an endohydrolase and a second strain expressing an exohydrolase, for cooperative degradation of a complex substrate. Our results suggest that there is a balance between increasing expression to enhance degradation versus the burden that higher expression causes. Once a threshold of burden is reached, the consortium will consistently perform better than an equivalent single-cell monoculture.ConclusionsWe demonstrate that resource-aware whole-cell models can be used to predict the benefits and limitations of using consortia systems to overcome burden. Our model predicts the region of expression where DOL would be beneficial for growth on starch, which will assist in making informed design choices for this, and other, complex-substrate degradation pathways.

Journal article

Bernier L, Stan G, Junier P, Stanley Cet al., 2022, Spores-on-a-chip: new frontiers for spore research, Trends in Microbiology, Vol: 30, Pages: 515-518, ISSN: 0966-842X

In recent years, microfluidic technologies have become widespread in biological science. However, the suitability of this technique for understanding different aspects of spore research has hardly been considered. Herein, we review recent developments in 'spores-on-a-chip' technologies, highlighting how they could be exploited to drive new frontiers in spore research.

Journal article

Sechkar K, Tuza ZA, Stan G-B, 2022, A linear programming-based strategy to save pipette tips in automates DNA assembly, SYNTHETIC BIOLOGY, Vol: 7

Journal article

Sechkar K, Tuza ZA, Stan G-B, 2022, A linear programming-based strategy to save pipette tips in automated DNA assembly, Synthetic Biology, ISSN: 2397-7000

Laboratory automation and mathematical optimization are key to improving the efficiency of synthetic biology research.While there are algorithms optimizing the construct designs and synthesis strategies for DNA assembly, the optimizationof how DNA assembly reaction mixes are prepared remains largely unexplored. Here, we focus on reducing the pipettetip consumption of a liquid-handling robot as it delivers DNA parts across a multi-well plate where several constructsare being assembled in parallel. We propose a linear programming formulation of this problem based on the capacitatedvehicle routing problem, as well as an algorithm which applies a linear programming solver to our formulation, henceproviding a strategy to prepare a given set of DNA assembly mixes using fewer pipette tips. The algorithm performedwell in randomly generated and real-life scenarios concerning several modular DNA assembly standards, proving capableof reducing the pipette tip consumption by up to 59% in large-scale cases. Combining automatic process optimizationand robotic liquid-handling, our strategy promises to greatly improve the efficiency of DNA assembly, either used aloneor combined with other algorithmic DNA assembly optimization methods.

Journal article

Climent-Catala A, Ouldridge TE, Stan G-BV, Bae Wet al., 2022, Building an RNA-based toggle switch using inhibitory RNA aptamers, ACS Synthetic Biology, Vol: 11, Pages: 562-569, ISSN: 2161-5063

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in degrading conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.

Journal article

Dwijayanti A, Storch M, Stan G-B, Baldwin GSet al., 2022, A modular RNA interference system for multiplexed gene regulation, Nucleic Acids Research, Vol: 50, ISSN: 0305-1048

The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.

Journal article

Slutsky I, Schratt G, Stan G-B, Nelson S, Bruggeman FJet al., 2021, Homeostasis, CELL SYSTEMS, Vol: 12, Pages: 1124-1126, ISSN: 2405-4712

Journal article

Csibra E, Stan G-B, 2021, FPCountR: Absolute protein quantification using fluorescence measurements

<jats:title>Abstract</jats:title><jats:p>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 a novel ‘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 600nm 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.</jats:p>

Journal article

Boo AR, Ledesma Amaro R, Stan G-B, 2021, Quorum sensing in synthetic biology: a review, Current Opinion in Systems Biology, Vol: 28, Pages: 1-14, ISSN: 2452-3100

In nature, quorum sensing is one of the mechanism bacterial populations use to communicate withtheir own species or across species to coordinate behaviours. For the last 20 years, synthetic biologistshave recognised the remarkable properties of quorum sensing to build genetic circuits responsive topopulation density. This has led to progress in designing dynamic, coordinated and sometimes multicellular systems for bio-production in metabolic engineering and for increased spatial and temporalcomplexity in synthetic biology. In this review, we highlight recent works focused on using quorumsensing to engineer cell-cell behaviour.

Journal article

Climent-Catala A, Ouldridge TE, Stan G-BV, Bae Wet al., 2021, Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

<jats:title>Abstract</jats:title><jats:p>Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.</jats:p><jats:sec><jats:title>Graphical TOC Entry</jats:title><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="468831v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Journal article

Perrino G, Hadjimitsis A, Ledesma Amaro R, Stan G-Bet al., 2021, Control engineering and synthetic biology: Working in synergy for the analysis and control of microbial systems, Current Opinion in Microbiology, Vol: 62, Pages: 68-75, ISSN: 1369-5274

The implementation of novel functionalities in living cells is a key aspect of synthetic biology. In the last decade, the field of synthetic biology has made progress working in synergy with control engineering, whose solid framework has provided concepts and tools to analyse biological systems and guide their design. In this review, we briefly highlight recent work focused on the application of control theoretical concepts and tools for the analysis and design of synthetic biology systems in microbial cells.

Journal article

Baig H, Fontanarossa P, Kulkarni V, McLaughlin J, Vaidyanathan P, Bartley B, Bhakta S, Bhatia S, Bissell M, Clancy K, Cox RS, Goñi Moreno A, Gorochowski T, Grunberg R, Lee J, Luna A, Madsen C, Misirli G, Nguyen T, Le Novere N, Palchick Z, Pocock M, Roehner N, Sauro H, Scott-Brown J, Sexton JT, Stan G-B, Tabor JJ, Terry L, Vazquez Vilar M, Voigt CA, Wipat A, Zong D, Zundel Z, Beal J, Myers Cet al., 2021, Synthetic biology open language visual (SBOL Visual) version 2.3, Journal of Integrative Bioinformatics, Vol: 18, ISSN: 1613-4516

People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.3 of SBOL Visual, which builds on the prior SBOL Visual 2.2 in several ways. First, the specification now includes higher-level "interactions with interactions," such as an inducer molecule stimulating a repression interaction. Second, binding with a nucleic acid backbone can be shown by overlapping glyphs, as with other molecular complexes. Finally, a new "unspecified interaction" glyph is added for visualizing interactions whose nature is unknown, the "insulator" glyph is deprecated in favor of a new "inert DNA spacer" glyph, and the polypeptide region glyph is recommended for showing 2A sequences.

Journal article

Plesa T, Stan G-B, Ouldridge TE, Bae Wet al., 2021, Quasi-robust control of biochemical reaction networks via stochastic morphing., Journal of the Royal Society Interface, Vol: 18, Pages: 1-14, ISSN: 1742-5662

One of the main objectives of synthetic biology is the development of molecular controllers that can manipulate the dynamics of a given biochemical network that is at most partially known. When integrated into smaller compartments, such as living or synthetic cells, controllers have to be calibrated to factor in the intrinsic noise. In this context, biochemical controllers put forward in the literature have focused on manipulating the mean (first moment) and reducing the variance (second moment) of the target molecular species. However, many critical biochemical processes are realized via higher-order moments, particularly the number and configuration of the probability distribution modes (maxima). To bridge the gap, we put forward the stochastic morpher controller that can, under suitable timescale separations, morph the probability distribution of the target molecular species into a predefined form. The morphing can be performed at a lower-resolution, allowing one to achieve desired multi-modality/multi-stability, and at a higher-resolution, allowing one to achieve arbitrary probability distributions. Properties of the controller, such as robustness and convergence, are rigorously established, and demonstrated on various examples. Also proposed is a blueprint for an experimental implementation of stochastic morpher.

Journal article

Cabello-Garcia J, Bae W, Stan G-BV, Ouldridge TEet al., 2021, Handhold-mediated strand displacement: a nucleic acid based mechanism for generating far-from-equilibrium assemblies through templated reactions., ACS Nano, Vol: 15, Pages: 3272-3283, ISSN: 1936-0851

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.

Journal article

Kuntz Nussio J, Thomas P, Stan G, Barahona Met al., 2021, Approximations of countably-infinite linear programs over bounded measure spaces, SIAM Journal on Optimization, Vol: 31, Pages: 604-625, ISSN: 1052-6234

We study a class of countably-infinite-dimensional linear programs (CILPs)whose feasible sets are bounded subsets of appropriately defined spaces ofmeasures. The optimal value, optimal points, and minimal points of these CILPscan be approximated by solving finite-dimensional linear programs. We show howto construct finite-dimensional programs that lead to approximations witheasy-to-evaluate error bounds, and we prove that the errors converge to zero asthe size of the finite-dimensional programs approaches that of the originalproblem. We discuss the use of our methods in the computation of the stationarydistributions, occupation measures, and exit distributions of Markov~chains.

Journal article

Kuntz J, Thomas P, Stan G-B, Barahona Met al., 2021, Stationary distributions of continuous-time Markov chains: a review of theory and truncation-based approximations, SIAM Review, ISSN: 0036-1445

Computing the stationary distributions of a continuous-time Markov chaininvolves solving a set of linear equations. In most cases of interest, thenumber of equations is infinite or too large, and cannot be solved analyticallyor numerically. Several approximation schemes overcome this issue by truncatingthe state space to a manageable size. In this review, we first give acomprehensive theoretical account of the stationary distributions and theirrelation to the long-term behaviour of the Markov chain, which is readilyaccessible to non-experts and free of irreducibility assumptions made instandard texts. We then review truncation-based approximation schemes payingparticular attention to their convergence and to the errors they introduce, andwe illustrate their performance with an example of a stochastic reactionnetwork of relevance in biology and chemistry. We conclude by elaborating oncomputational trade-offs associated with error control and some open questions.

Journal article

Selles Vidal L, Ayala R, Stan G-B, Ledesma-Amaro Ret al., 2021, rfaRm: An R client-side interface to facilitate the analysis of the Rfam database of RNA families, PLoS One, Vol: 16, ISSN: 1932-6203

rfaRm is an R package providing a client-side interface for the Rfam database of non-coding RNA and other structured RNA elements. The package facilitates the search of the Rfam database by keywords or sequences, as well as the retrieval of all available information about specific Rfam families, such as member sequences, multiple sequence alignments, secondary structures and covariance models. By providing such programmatic access to the Rfam database, rfaRm enables genomic workflows to incorporate information about non-coding RNA, whose potential cannot be fully exploited just through interactive access to the database. The features of rfaRm are demonstrated by using it to analyze the SARS-CoV-2 genome as an example case.

Journal article

Sarvari P, Ingram D, Stan G-B, 2021, A modelling framework linking resource-based stochastic translation to the optimal design of synthetic constructs, Biology, Vol: 10, ISSN: 2079-7737

The effect of gene expression burden on engineered cells has motivated the use of “whole-cell models” (WCMs) that use shared cellular resources to predict how unnatural gene expression affects cell growth. A common problem with many WCMs is their inability to capture translation in sufficient detail to consider the impact of ribosomal queue formation on mRNA transcripts. To address this, we have built a “stochastic cell calculator” (StoCellAtor) that combines a modified TASEP with a stochastic implementation of an existing WCM. We show how our framework can be used to link a synthetic construct’s modular design (promoter, ribosome binding site (RBS) and codon composition) to protein yield during continuous culture, with a particular focus on the effects of low-efficiency codons and their impact on ribosomal queues. Through our analysis, we recover design principles previously established in our work on burden-sensing strategies, namely that changing promoter strength is often a more efficient way to increase protein yield than RBS strength. Importantly, however, we show how these design implications can change depending on both the duration of protein expression, and on the presence of ribosomal queues.

Journal article

Ouldridge T, Stan G-B, Bae W, 2020, In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 21, Pages: 265-271, ISSN: 1530-6984

Synthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components, namely, multistranded complexes known as gates, in situ, i.e., in living cells or other autonomous systems. Here, we propose the use of naturally occurring self-cleaving ribozymes to cut a single-stranded RNA transcript into a gate complex of shorter strands, thereby opening new possibilities for the autonomous and continuous production of RNA strands in a stoichiometrically and structurally controlled way.

Journal article

Frei T, Cella F, Tedeschi F, Gutiérrez J, Stan G-B, Khammash M, Siciliano Vet al., 2020, Characterization and mitigation of gene expression burden in mammalian cells, Nature Communications, Vol: 11, ISSN: 2041-1723

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.

Journal article

Baig H, Fontanarrosa P, Kulkarni V, McLaughlin J, Vaidyanathan P, Bartley B, Bhatia S, Bhakta S, Bissell M, Clancy K, Cox RS, Moreno AG, Gorochowski T, Grunberg R, Luna A, Madsen C, Misirli G, Nguyen T, Le Novere N, Palchick Z, Pocock M, Roehner N, Sauro H, Scott-Brown J, Sexton JT, Stan G-B, Tabor JJ, Vilar MV, Voigt CA, Wipat A, Zong D, Zundel Z, Beal J, Myers Cet al., 2020, Synthetic biology open language visual (SBOL visual) version 2.2, Journal of Integrative Bioinformatics, Vol: 17, Pages: 1-85, ISSN: 1613-4516

People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.2 of SBOL Visual, which builds on the prior SBOL Visual 2.1 in several ways. First, the grounding of molecular species glyphs is changed from BioPAX to SBO, aligning with the use of SBO terms for interaction glyphs. Second, new glyphs are added for proteins, introns, and polypeptide regions (e. g., protein domains), the prior recommended macromolecule glyph is deprecated in favor of its alternative, and small polygons are introduced as alternative glyphs for simple chemicals.

Journal article

Misirli G, Beal J, Gorochowski TE, Stan G-B, Wipat A, Myers CJet al., 2020, SBOL visual 2 ontology, ACS Synthetic Biology, Vol: 9, Pages: 972-977, ISSN: 2161-5063

Standardizing the visual representation of genetic parts and circuits is essential for unambiguously creating and interpreting genetic designs. To this end, an increasing number of tools are adopting well-defined glyphs from the Synthetic Biology Open Language (SBOL) Visual standard to represent various genetic parts and their relationships. However, the implementation and maintenance of the relationships between biological elements or concepts and their associated glyphs has up to now been left up to tool developers. We address this need with the SBOL Visual 2 Ontology, a machine-accessible resource that provides rules for mapping from genetic parts, molecules, and interactions between them, to agreed SBOL Visual glyphs. This resource, together with a web service, can be used as a library to simplify the development of visualization tools, as a stand-alone resource to computationally search for suitable glyphs, and to help facilitate integration with existing biological ontologies and standards in synthetic biology.

Journal article

Misirli G, Beal J, Gorochowski TE, Stan G-B, Wipat A, Myers Cet al., 2020, SBOL Visual 2 Ontology, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>Standardising the visual representation of genetic parts and circuits is vital for unambiguously creating and interpreting genetic designs. To this end, an increasing number of tools are adopting well-defined glyphs from the Synthetic Biology Open Language (SBOL) Visual standard to represent various genetic parts and their relationships. However, the implementation and maintenance of the relationships between biological elements or concepts and their associated glyphs has to now been left up to tool developers. We address this need with the SBOL Visual 2 Ontology, a machine-accessible resource that provides rules for mapping from genetic parts, molecules, and interactions between them, to agreed SBOL Visual glyphs. This resource, together with a web service, can be used as a library to simplify the development of visualization tools, as a stand-alone resource to computationally search for suitable glyphs, and to help facilitate integration with existing biological ontologies and standards in synthetic biology.</jats:p><jats:sec><jats:title>Graphical TOC Entry</jats:title><jats:fig id="ufig1" position="anchor" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="918417v3_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Working paper

Sootla A, Stan G-B, Ernst D, 2020, Solving Optimal Control Problems for Monotone Systems Using the Koopman Operator, Lecture Notes in Control and Information Sciences, Publisher: Springer International Publishing, Pages: 283-312, ISBN: 9783030357122

© Springer Nature Switzerland AG 2020. Koopman operator theory offers numerous techniques for analysis and control of complex systems. In particular, in this chapter we will argue that for the problem of convergence to an equilibrium, the Koopman operator can be used to take advantage of the geometric properties of controlled systems, thus making the optimal solutions more transparent, and easier to analyze and implement. The motivation for the study of the convergence problem comes from biological applications, where easy-to-implement and easy-to-analyze solutions are of particular value. At the moment, theoretical results have been developed for a class of nonlinear systems called monotone systems. However, the core ideas presented here can be applied heuristically to non-monotone systems. Furthermore, the convergence problem can serve as a building block for solving other control problems such as switching between stable equilibria or inducing oscillations. These applications are illustrated in biologically inspired numerical examples.

Book chapter

Kuntz Nussio J, Thomas P, Stan GB, Barahona Met al., 2019, Bounding the stationary distributions of the chemical master equation via mathematical programming, Journal of Chemical Physics, Vol: 151, ISSN: 0021-9606

The stochastic dynamics of biochemical networks are usually modelled with the chemical master equation (CME). The stationary distributions of CMEs are seldom solvable analytically, and numerical methods typically produce estimates with uncontrolled errors. Here, we introduce mathematical programming approaches that yield approximations of these distributions with computable error bounds which enable the verification of their accuracy. First, we use semidefinite programming to compute increasingly tighter upper and lower bounds on the moments of the stationary distributions for networks with rational propensities. Second, we use these moment bounds to formulate linear programs that yield convergent upper and lower bounds on the stationary distributions themselves, their marginals and stationary averages. The bounds obtained also provide a computational test for the uniqueness of the distribution. In the unique case, the bounds form an approximation of the stationary distribution with a computable bound on its error. In the non unique case, our approach yields converging approximations of the ergodic distributions. We illustrate our methodology through several biochemical examples taken from the literature: Schl¨ogl’s model for a chemical bifurcation, a two-dimensional toggle switch, a model for bursty gene expression, and a dimerisation model with multiple stationary distributions.

Journal article

Haines M, Storch M, Oyarzun D, Stan G, Baldwin Get al., 2019, Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR), Synthetic Biology, Vol: 4, Pages: 1-10, ISSN: 2397-7000

In vitro selection of ligand-responsive ribozymes can identify rare, functional sequences from large libraries. While powerful, key caveats of this approach include lengthy and demanding experimental workflows; unpredictable experimental outcomes and unknown functionality of enriched sequences in vivo. To address the first of these limitations we developed Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR). LigASERR is scalable, amenable to automation and requires less time to implement compared to alternative methods. To improve the predictability of experiments, we modelled the underlying selection process, predicting experimental outcomes based on sequence and population parameters. We applied this new methodology and model to the enrichment of a known, in vitro-selected sequence from a bespoke library. Prior to implementing selection, conditions were optimised and target sequence dynamics accurately predicted for the majority of the experiment. In addition to enriching the target sequence, we identified two new, theophylline-activated ribozymes. Notably, all three sequences yielded riboswitches functional in Escherichia coli, suggesting LigASERR and similar in vitro selection methods can be utilised for generating functional riboswitches in this organism.

Journal article

Madsen C, Goni Moreno A, Palchick Z P U, Roehner N, Bartley B, Bhatia S, Bhakta S, Bissell M, Clancy K, Cox RS, Gorochowski T, Grunberg R, Luna A, McLaughlin J, Nguyen T, Le Novere N, Pocock M, Sauro H, Scott-Brown J, Sexton JT, Stan G-B, Tabor JJ, Voigt CA, Zundel Z, Myers C, Beal J, Wipat Aet al., 2019, Synthetic Biology Open Language Visual (SBOL Visual) version 2.1, Journal of Integrative Bioinformatics, Vol: 16, Pages: 1-78, ISSN: 1613-4516

People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species . Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.1 of SBOL Visual, which builds on the prior SBOL Visual 2.0 standard by expanding diagram syntax to include methods for showing modular structure and mappings between elements of a system, interactions arrows that can split or join (with the glyph at the split or join indicating either superposition or a chemical process), and adding new glyphs for indicating genomic context (e.g., integration into a plasmid or genome) and for stop codons.

Journal article

Boo A, Ellis T, Stan G, 2019, Host-aware synthetic biology, Current Opinion in Systems Biology, Vol: 14, Pages: 66-72, ISSN: 2452-3100

Unnatural gene expression imposes a load on engineered microorganisms thatdecreases their growth and subsequent production yields, a phenomenon knownasburden. In the last decade, the field of synthetic biology has made progress onthe development of biomolecular feedback control systems and other approachesthat can improve the growth of engineered cells, as well as the genetic stability,portability and robust performance of cell-hosted synthetic constructs. In thisreview, we highlight recent work focused on the development of host-aware syn-thetic biology.

Journal article

Kuntz J, Thomas P, Stan G-B, Barahona Met al., 2019, The exit time finite state projection scheme: bounding exit distributions and occupation measures of continuous-time Markov chains, SIAM Journal on Scientific Computing, Vol: 41, Pages: A748-A769, ISSN: 1064-8275

We introduce the exit time finite state projection (ETFSP) scheme, a truncation- based method that yields approximations to the exit distribution and occupation measure associated with the time of exit from a domain (i.e., the time of first passage to the complement of the domain) of time-homogeneous continuous-time Markov chains. We prove that: (i) the computed approximations bound the measures from below; (ii) the total variation distances between the approximations and the measures decrease monotonically as states are added to the truncation; and (iii) the scheme converges, in the sense that, as the truncation tends to the entire state space, the total variation distances tend to zero. Furthermore, we give a computable bound on the total variation distance between the exit distribution and its approximation, and we delineate the cases in which the bound is sharp. We also revisit the related finite state projection scheme and give a comprehensive account of its theoretical properties. We demonstrate the use of the ETFSP scheme by applying it to two biological examples: the computation of the first passage time associated with the expression of a gene, and the fixation times of competing species subject to demographic noise.

Journal article

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