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Research

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.

Publications

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Journal article
Poole W, Ouldridge T, Gopalkrishnan M,
Autonomous learning of generative models with chemical reaction network ensembles
, Journal of the Royal Society Interface, ISSN: 1742-5662
Can a micron sized sack of interacting molecules autonomously learn an internalmodel of a complex and fluctuating environment? We draw insights from controltheory, machine learning theory, chemical reaction network theory, and statisticalphysics to develop a general architecture whereby a broad class of chemical systemscan autonomously learn complex distributions. Our construction takes the form ofa chemical implementation of machine learning’s optimization workhorse: gradientdescent on the relative entropy cost function which we demonstrate can be viewedas a form of integral feedback control. We show how this method can be applied tooptimize any detailed balanced chemical reaction network and that the constructionis capable of using hidden units to learn complex distributions.
Journal article
Smith F, Goetz J, Jurinovic K, Stevens M, Ouldridge Tet al., 2024,
Strong sequence-dependence in RNA/DNA hybrid strand displacement kinetics
, Nanoscale, Vol: 16, Pages: 17624-17637, ISSN: 2040-3364
Strand displacement reactions underlie dynamic nucleic acid nanotechnology. The kinetic and thermodynamic features of DNA-based displacement reactions are well understood and well predicted by current computational models. By contrast, understanding of RNA/DNA hybrid strand displacement kinetics is limited, restricting the design of increasingly complex RNA/DNA hybrid reaction networks with more tightly regulated dynamics. Given the importance of RNA as a diagnostic biomarker, and its critical role in intracellular processes,this shortfall is particularly limiting for the development of strand displacement-based therapeutics and diagnostics. Herein, we characterise 22 RNA/DNA hybrid strand displacement systems, alongside 11 DNA/DNA systems, varying a range of common design parameters including toehold length and branch migration domain length. We observe the differences in stability between RNA-DNA hybrids and DNA-DNA duplexes have large effects on strand displacement rates, with rates for equivalent sequences differing by up to 3 orders of magnitude. Crucially, however, this effect is strongly sequence-dependent, with RNA invaders strongly favoured in a system with RNA strands of high purine content, and disfavoured in a system when the RNA strands have low purine content. These results lay the groundwork for more general design principles, allowing for creation of de novo reaction networks with novel complexity while maintaining predictable reaction kinetics.
Journal article
Mukherjee R, Sengar A, Cabello Garcia J, Ouldridge Tet al., 2024,
Kinetic proofreading can enhance specificity in a non-enzymatic DNA strand displacement network
, Journal of the American Chemical Society, Vol: 146, Pages: 18916-18926, ISSN: 0002-7863
Kinetic proofreading is used throughout natural systems to enhance the specificity of molecular recognition. At its most basic level, kinetic proofreading uses a supply of chemical fuel to drive a recognition interaction out of equilibrium, allowing a single free-energy difference between correct and incorrect targets to be exploited two or more times. Despite its importance in biology, there has been little effort to incorporate kinetic proofreading into synthetic systems in which molecular recognition is important, such as nucleic acid nanotechnology. In this article, we introduce a DNA strand displacement-based kinetic proofreading motif, showing that the consumption of a DNA-based fuel can be used to enhance molecular recognition during a templated dimeri zation reaction. We then show that kinetic proofreading can enhance the specificity with which a probe discriminates single nucleo tide mutations, both in terms of the initial rate with which the probe reacts and the long-time behaviour.
Journal article
Climent-Catala A, Casas-Rodrigo I, Iyer S, Ledesma-Amaro R, Ouldridge TEet al., 2023,
Evaluating DFHBI-responsive RNA light-up aptamers as fluorescent reporters for gene expression
, ACS Synthetic Biology, Vol: 12, Pages: 3754-3765, ISSN: 2161-5063
Protein-based fluorescent reporters have been widely used to characterize and localize biological processes in living cells. However, these reporters may have certain drawbacks for some applications, such as transcription-based studies or biological interactions with fast dynamics. In this context, RNA nanotechnology has emerged as a promising alternative, suggesting the use of functional RNA molecules as transcriptional fluorescent reporters. RNA-based aptamers can bind to nonfluorescent small molecules to activate their fluorescence. However, their performance as reporters of gene expression in living cells has not been fully characterized, unlike protein-based reporters. Here, we investigate the performance of three RNA light-up aptamers─F30-2xdBroccoli, tRNA-Spinach, and Tornado Broccoli─as fluorescent reporters for gene expression in Escherichia coli and compare them to a protein reporter. We examine the activation range and effect on the cell growth of RNA light-up aptamers in time-course experiments and demonstrate that these aptamers are suitable transcriptional reporters over time. Using flow cytometry, we compare the variability at the single-cell level caused by the RNA fluorescent reporters and protein-based reporters. We found that the expression of RNA light-up aptamers produced higher variability in a population than that of their protein counterpart. Finally, we compare the dynamical behavior of these RNA light-up aptamers and protein-based reporters. We observed that RNA light-up aptamers might offer faster dynamics compared to a fluorescent protein in E. coli. The implementation of these transcriptional reporters may facilitate transcription-based studies, gain further insights into transcriptional processes, and expand the implementation of RNA-based circuits in bacterial cells.
Journal article
Plesa T, Dack A, Ouldridge T, 2023,
Integral feedback in synthetic biology: negative-equilibrium catastrophe
, Journal of Mathematical Chemistry, Vol: 61, Pages: 1980-2018, ISSN: 0259-9791
A central goal of synthetic biology is the design of molecular controllers that can manipulate the dynamics of intracellular networks in a stable and accurate manner. To address the factthat detailed knowledge about intracellular networks is unavailable, integral-feedback controllers(IFCs) have been put forward for controlling molecular abundances. These controllers can maintainaccuracy in spite of the uncertainties in the controlled networks. However, this desirable feature isachieved only if stability is also maintained. In this paper, we show that molecular IFCs can sufferfrom a hazardous instability called negative-equilibrium catastrophe (NEC), whereby all nonnegative equilibria vanish under the action of the controllers, and some of the molecular abundancesblow up. We show that unimolecular IFCs do not exist due to a NEC. We then derive a familyof bimolecular IFCs that are safeguarded against NECs when uncertain unimolecular networks,with any number of molecular species, are controlled. However, when IFCs are applied on uncertain bimolecular (and hence most intracellular) networks, we show that preventing NECs generallybecomes an intractable problem as the number of interacting molecular species increases. NECstherefore place a fundamental limit to design and control of molecular networks.
Journal article
Cella F, Perrino G, Tedeschi F, Viero G, Bosia C, Stan G-B, Siciliano Vet al., 2023,
MIRELLA: a mathematical model explains the effect of microRNA-mediated synthetic genes regulation on intracellular resource allocation
, Nucleic Acids Research, Vol: 51, Pages: 3452-3464, ISSN: 0305-1048
Competition for intracellular resources, also known as gene expression burden, induces coupling between independently co-expressed genes, a detrimental effect on predictability and reliability of gene circuits in mammalian cells. We recently showed that microRNA (miRNA)-mediated target downregulation correlates with the upregulation of a co-expressed gene, and by exploiting miRNAs-based incoherent-feed-forward loops (iFFLs) we stabilise a gene of interest against burden. Considering these findings, we speculate that miRNA-mediated gene downregulation causes cellular resource redistribution. Despite the extensive use of miRNA in synthetic circuits regulation, this indirect effect was never reported before. Here we developed a synthetic genetic system that embeds miRNA regulation, and a mathematical model, MIRELLA, to unravel the miRNA (MI) RolE on intracellular resource aLLocAtion. We report that the link between miRNA-gene downregulation and independent genes upregulation is a result of the concerted action of ribosome redistribution and ‘queueing-effect’ on the RNA degradation pathway. Taken together, our results provide for the first time insights into the hidden regulatory interaction of miRNA-based synthetic networks, potentially relevant also in endogenous gene regulation. Our observations allow to define rules for complexity- and context-aware design of genetic circuits, in which transgenes co-expression can be modulated by tuning resource availability via number and location of miRNA target sites.
Journal article
Qureshi BJ, Juritz J, Poulton JM, Beersing-Vasquez A, Ouldridge TEet al., 2023,
A universal method for analyzing copolymer growth
, Journal of Chemical Physics, Vol: 158, Pages: 1-22, ISSN: 0021-9606
Polymers consisting of more than one type of monomer, known as copolymers,are vital to both living and synthetic systems. Copolymerisation has beenstudied theoretically in a number of contexts, often by considering a Markovprocess in which monomers are added or removed from the growing tip of a longcopolymer. To date, the analysis of the most general models of this class hasnecessitated simulation. We present a general method for analysing suchprocesses without resorting to simulation. Our method can be applied to modelswith an arbitrary network of sub-steps prior to addition or removal of amonomer, including non-equilibrium kinetic proofreading cycles. Moreover, theapproach allows for a dependency of addition and removal reactions on theneighbouring site in the copolymer, and thermodynamically self-consistentmodels in which all steps are assumed to be microscopically reversible. Usingour approach, thermodynamic quantities such as chemical work; kineticquantities such as time taken to grow; and statistical quantities such as thedistribution of monomer types in the growing copolymer can be derived eitheranalytically or numerically directly from the model definition.
Journal article
Csibra E, Stan G-B, 2022,
Absolute protein quantification using fluorescence measurements with FPCountR
, Nature Communications, Vol: 40, ISSN: 2041-1723
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.
Journal article
Ouldridge T, Hertel S, Spinney R, Xu S, Morris R, Lee Let al., 2022,
The stability and number of nucleating interactions determine DNA hybridisation rates in the absence of secondary structure
, Nucleic Acids Research, Vol: 50, Pages: 7829-7841, ISSN: 0305-1048
The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies.However, the precise physical mechanisms that determine why different DNA sequences hybridise at differentrates are not well understood. Secondary structure is one predictable factor that influences hybridisation ratesbut is not sufficient on its own to fully explain the observed sequence-dependent variance. In this context, wemeasured hybridisation rates of 43 different DNA sequences that are not predicted to form secondarystructure and present a parsimonious physically justified model to quantify our observations. Accounting onlyfor the combinatorics of complementary nucleating interactions and their sequence-dependent stability, themodel achieves good correlation with experiment with only two free parameters. Our results indicate thatgreater repetition of Watson-Crick pairs increases the number of initial states able to proceed to fullhybridisation, with the stability of those pairings dictating the likelihood of such progression, thus providingnew insight into the physical factors underpinning DNA hybridisation rates.
Journal article
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
Beardall WAV, Stan G-B, Dunlop MJ, 2022,
Deep Learning Concepts and Applications for Synthetic Biology.
, GEN Biotechnology, Vol: 1, Pages: 360-371, ISSN: 2768-1556
Synthetic biology has a natural synergy with deep learning. It can be used to generate large data sets to train models, for example by using DNA synthesis, and deep learning models can be used to inform design, such as by generating novel parts or suggesting optimal experiments to conduct. Recently, research at the interface of engineering biology and deep learning has highlighted this potential through successes including the design of novel biological parts, protein structure prediction, automated analysis of microscopy data, optimal experimental design, and biomolecular implementations of artificial neural networks. In this review, we present an overview of synthetic biology-relevant classes of data and deep learning architectures. We also highlight emerging studies in synthetic biology that capitalize on deep learning to enable novel understanding and design, and discuss challenges and future opportunities in this space.
Journal article
Webb A, Allan F, Kelwick R, Beshah F, Kinunghi S, Templeton MR, Emery A, Freemont Pet al., 2022,
Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS
, PLOS Neglected Tropical Diseases, Vol: 16, Pages: 1-19, ISSN: 1935-2727
Schistosomiasis, also known as bilharzia or snail fever, is a debilitating neglected tropical disease (NTD), caused by parasitic trematode flatworms of the genus Schistosoma, that has an annual mortality rate of 280,000 people in sub-Saharan Africa alone. Schistosomiasis is transmitted via contact with water bodies that are home to the intermediate host snail which shed the infective cercariae into the water. Schistosome lifecycles are complex, and while not all schistosome species cause human disease, endemic regions also typically feature animal infecting schistosomes that can have broader economic and/or food security implications. Therefore, the development of species-specific Schistosoma detection technologies may help to inform evidence-based local environmental, food security and health systems policy making. Crucially, schistosomiasis disproportionally affects low- and middle-income (LMIC) countries and for that reason, environmental screening of water bodies for schistosomes may aid with the targeting of water, sanitation, and hygiene (WASH) interventions and preventive chemotherapy to regions at highest risk of schistosomiasis transmission, and to monitor the effectiveness of such interventions at reducing the risk over time. To this end, we developed a DNA-based biosensor termed Specific Nucleic AcId Ligation for the detection of Schistosomes or ‘SNAILS’. Here we show that ‘SNAILS’ enables species-specific detection from genomic DNA (gDNA) samples that were collected from the field in endemic areas.
Book chapter
Ouldridge T, Doye J, Louis A, Schreck J, Romano F, Harrison R, Mosayebi M, Engel Met al., 2022,
Free energy landscapes of DNA and its assemblies: perspectives from coarse-grained modelling
, Energy Landscapes of Nanoscale Systems, Publisher: Elsevier, Pages: 195-210, ISBN: 9780128244067
This chapter will provide an overview of how characterising free energy landscapes can provide insights into the biophysical properties of DNA, as well as into the behaviour of the DNA assemblies used in the field of DNA nanotechnology. The landscapes for these complex systems are accessible through the use of accurate coarse-grained descriptions of DNA. Particular foci will be the landscapes associated with DNA self-assembly and mechanical deformation, where the latter can arise from either externally imposed forces or internal stresses.
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 automated DNA assembly
, Synthetic Biology, Vol: 7, Pages: 1-8, 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
Juritz J, Poulton JM, Ouldridge TE, 2022,
Minimal mechanism for cyclic templating of length-controlled copolymers under isothermal conditions
, Journal of Chemical Physics, Vol: 156, ISSN: 0021-9606
The production of sequence-specific copolymers using copolymer templates is fundamental to the synthesis of complex biological molecules and is a promising framework for the synthesis of synthetic chemical complexes. Unlike the superficially similar process of self-assembly, however, the development of synthetic systems that implement templated copying of copolymers under constant environmental conditions has been challenging. The main difficulty has been overcoming product inhibition or the tendency of products to adhere strongly to their templates—an effect that gets exponentially stronger with the template length. We develop coarse-grained models of copolymerization on a finite-length template and analyze them through stochastic simulation. We use these models first to demonstrate that product inhibition prevents reliable template copying and then ask how this problem can be overcome to achieve cyclic production of polymer copies of the right length and sequence in an autonomous and chemically driven context. We find that a simple addition to the model is sufficient to generate far longer polymer products that initially form on, and then separate from, the template. In this approach, some of the free energy of polymerization is diverted into disrupting copy–template bonds behind the leading edge of the growing copy copolymer. By additionally weakening the final copy–template bond at the end of the template, the model predicts that reliable copying with a high yield of full-length, sequence-matched products is possible over large ranges of parameter space, opening the way to the engineering of synthetic copying systems that operate autonomously.
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
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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.

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