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Synthetic Biology underpins advances in the bioeconomy

Biological systems - including the simplest cells - exhibit a broad range of functions to thrive in their environment. Research in the Imperial College Centre for Synthetic Biology is focused on the possibility of engineering the underlying biochemical processes to solve many of the challenges facing society, from healthcare to sustainable energy. In particular, we model, analyse, design and build biological and biochemical systems in living cells and/or in cell extracts, both exploring and enhancing the engineering potential of biology. 

As part of our research we develop novel methods to accelerate the celebrated Design-Build-Test-Learn synthetic biology cycle. As such research in the Centre for Synthetic Biology highly multi- and interdisciplinary covering computational modelling and machine learning approaches; automated platform development and genetic circuit engineering ; multi-cellular and multi-organismal interactions, including gene drive and genome engineering; metabolic engineering; in vitro/cell-free synthetic biology; engineered phages and directed evolution; and biomimetics, biomaterials and biological engineering.

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  • Journal article
    Liu H, Hong F, Smith F, Goertz J, Ouldridge T, Stevens MM, Yan H, Šulc Pet al., 2021,

    Kinetics of RNA and RNA:DNA hybrid strand displacement

    , ACS Synthetic Biology, Vol: 10, Pages: 3066-3073, ISSN: 2161-5063

    In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

  • Journal article
    Boo AR, Ledesma Amaro R, Stan G-B,

    Quorum sensing in synthetic biology: a review

    , Current Opinion in Systems Biology, 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
    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
    Poulton JM, Ouldridge TE, 2021,

    Edge-effects dominate copying thermodynamics for finite-length molecular oligomers

    , New Journal of Physics, Vol: 23, Pages: 1-14, ISSN: 1367-2630

    A signature feature of living systems is their ability to produce copies ofinformation-carrying molecular templates such as DNA. These copies are madeby assembling a set of monomer molecules into a linear macromolecule with a sequence determined by the template. The copies produced have a finite length –they are often “oligomers”, or short polymers – and must eventually detach fromtheir template. We explore the role of the resultant initiation and termination ofthe copy process in the thermodynamics of copying. By splitting the free-energychange of copy formation into informational and chemical terms, we show that,surprisingly, copy accuracy plays no direct role in the overall thermodynamics. Instead, finite-length templates function as highly-selective engines that interconvertchemical and information-based free energy stored in the environment; it is thermodynamically costly to produce outputs that are more similar to the oligomersin the environment than sequences obtained by randomly sampling monomers. Incontrast to previous work that neglects separation, any excess free energy stored incorrelations between copy and template sequences is lost when the copy fully detaches and mixes with the environment; these correlations therefore do not featurein the overall thermodynamics. Previously-derived constraints on copy accuracytherefore only manifest as kinetic barriers experienced while the copy is templateattached; these barriers are easily surmounted by shorter oligomers.

  • 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
    Sengar A, Ouldridge TE, Henrich O, Rovigatti L, Sulc Pet al., 2021,

    A primer on the oxDNA model of DNA: When to use it, how to simulate it and how to interpret the results

    , Frontiers in Molecular Biosciences, Vol: 8, Pages: 1-22, ISSN: 2296-889X

    The oxDNA model of DNA has been applied widely to systems in biology,biophysics and nanotechnology. It is currently available via two independentopen source packages. Here we present a set of clearly-documented exemplarsimulations that simultaneously provide both an introduction to simulating themodel, and a review of the model's fundamental properties. We outline howsimulation results can be interpreted in terms of -- and feed into ourunderstanding of -- less detailed models that operate at larger length scales,and provide guidance on whether simulating a system with oxDNA is worthwhile.

  • Journal article
    Ferdous Z, Fuchs S, Behrends V, Trasanidis N, Vlachou D, Christophides GKet al., 2021,

    Anopheles coluzziistearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding

    , PLoS Pathogens, Vol: 17, ISSN: 1553-7366

    Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne

  • Journal article
    Gilbert C, Tang T-C, Ott W, Dorr B, Shaw W, Sun G, Lu T, Ellis Tet al., 2021,

    Living materials with programmable functionalities grown from engineered microbial co-cultures

    , Nature Materials, Vol: 20, Pages: 691-700, ISSN: 1476-1122

    Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically-engineered organisms. Here we describe an approach to fabricate functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulosed-based engineered living materials with potential applications in biosensing and biocatalysis.

  • Journal article
    Miyano T, Tanaka R, 2021,

    Identification of keratinocyte subpopulations in transcriptome to evaluate drug effects in atopic dermatitis

    , British Journal of Dermatology, Vol: 184, Pages: 798-799, ISSN: 0007-0963
  • 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
    Elani Y, 2021,

    Interfacing living and synthetic cells as an emerging frontier in synthetic biology

    , Angewandte Chemie International Edition, Vol: 60, Pages: 5602-5611, ISSN: 1433-7851

    The construction of artificial cells from inanimate molecular building blocks is one of the grand challenges of our time. In addition to being used as simplified cell models to decipher the rules of life, artificial cells have the potential to be designed as micromachines deployed in a host of clinical and industrial applications. The attractions of engineering artificial cells from scratch, as opposed to re‐engineering living biological cells, are varied. However, it is clear that artificial cells cannot currently match the power and behavioural sophistication of their biological counterparts. Given this, many in the synthetic biology community have started to ask: is it possible to interface biological and artificial cells together to create hybrid living/synthetic systems that leverage the advantages of both? This article will discuss the motivation behind this cellular bionics approach, in which the boundaries between living and non‐living matter are blurred by bridging top‐down and bottom‐up synthetic biology. It details the state of play of this nascent field and introduces three generalised hybridisation modes that have emerged.

  • Journal article
    Nousbeck J, McAleer MA, Hurault G, Kenny E, Harte K, Kezic S, Tanaka RJ, Irvine ADet al., 2021,

    miRNA analysis of childhood atopic dermatitis reveals a role for miR-451a

    , British Journal of Dermatology, Vol: 184, Pages: 514-523, ISSN: 0007-0963

    BACKGROUND: MicroRNAs (miRNAs), important regulators of gene expression, have been implicated in a variety of disorders. The expression pattern of miRNAs in pediatric atopic dermatitis (AD) has not been well studied. OBJECTIVE: We sought to investigate miRNA expression profiles in different blood compartments of infants with AD. METHODS: Small RNA and HTG-Edge sequencing were performed to identify differentially expressed miRNAs in PBMCs and plasma of AD infants versus age-matched healthy controls, with reverse transcription quantitative real-time PCR used for validation and measurement of miRNA targets. Logistic regression models with AUROC estimation was used to evaluate the diagnostic potential of chosen miRNAs for AD. RESULTS: RNA sequencing was performed to access miRNA expression profile in pediatric AD. We identified ten differentially expressed miRNAs in PBMCs and eight dysregulated miRNAs in plasma of AD infants compared to controls. Upregulated miRNAs in PBMCs included miRNAs known to be involved in inflammation: miR-223-3p, miR-126-5p and miR-143-3p. Differential expression of only one miRNA, miR-451a, was observed in both PBMCs and plasma of children with AD. Dysregulation of three miRNAs: miR-451a, miR-143-3p and miR-223-3p was validated in larger number of samples and miR-451a was identified as a predictive biomarker for the early diagnosis of the disease. Experimentally verified targets of miR-451a, IL6R and PSMB8, were increased in AD patients, negatively correlated with miR-451a levels and upregulated following inhibition of miR-451a in PBMCs. CONCLUSION: In infants with AD, a distinct peripheral blood miRNA signature is seen, highlighting the systemic effects of the disease. miR-451a is uniquely expressed in different blood compartments of AD patients and may serve as a promising novel biomarker for the early diagnosis of AD.

  • Journal article
    Aw R, De Wachter C, Laukens B, De Rycke R, De Bruyne M, Bell D, Callewaert N, Polizzi KMet al., 2021,

    Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

    , Traffic, Vol: 22, Pages: 48-63, ISSN: 1398-9219

    The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.

  • Journal article
    Zorzan I, López AR, Malyshava A, Ellis T, Barberis Met al., 2021,

    Synthetic designs regulating cellular transitions: Fine-tuning of switches and oscillators

    , Current Opinion in Systems Biology, Vol: 25, Pages: 11-26

    Biological circuits are responsible for transitions between cellular states in a timely fashion. For example, stem cells switch from an undifferentiated (unstable) state to a differentiated (stable) state. Conversely, cell cycle and circadian clocks are completed through transitions among successive (stable) states, i.e. waves, with (unstable) states switching them at definite timing. These transitions irreversibly determine the biological response or fate of a cell, to commit to reversible switches or to generate periodic oscillations of its state. Here, we review synthetic circuits that, in silico and in vivo, allow a cell to ‘make a decision’, i.e. to select which state to reach, among multiple ones available, through definite network designs. Specifically, we propose and discuss the designs, and their constituents motifs, which we consider to be more prone to reprogram cell behaviour, and whose parameters can be fine-tuned through systems biology and tested experimentally through Synthetic Biology. For these designs, exploration of the parameter space and of the influence of (external) cellular signals – which modulate circuit parameters – allows for the prediction of the circuit's response and its consequent impact on cell fate.

  • Journal article
    Liberante FG, Ellis T, 2021,

    From kilobases to megabases: Design and delivery of large DNA constructs into mammalian genomes

    , Current Opinion in Systems Biology, Vol: 25, Pages: 1-10

    As DNA synthesis has become cheaper, it has made assembly of larger and larger genes possible. To fully realise this opportunity for a new era of synthetic biology in mammals, a number of gaps are beginning to be addressed in the design, synthesis, assembly and delivery of DNA constructs into large genomes. While current DNA design software is still inadequate for complex mammalian genomes, editing large bacterial artificial chromosomes is now easier. Newer viral technologies, such as herpes simplex virus, have been adapted for assembly of mammalian artificial chromosomes. Further advances in genome engineering, such as CRISPR- and Retron-based systems, will simplify targeted insertion of big DNA – all promising an exciting future for this field.

  • 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 R, Vidal LS, 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.

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