81 results found
Gowers G, Chee S, Bell D, et al., 2020, Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening, Nature Communications, Vol: 11, ISSN: 2041-1723
Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.
Gilbert C, Tang T-C, Ott W, et al., 2019, Living materials with programmable functionalities grown from engineered microbial co-cultures
<jats:title>ABSTRACT</jats:title><jats:p>Biological systems assemble tissues and structures with advanced properties in ways that cannot be achieved by man-made materials. Living materials self-assemble under mild conditions, are autonomously patterned, can self-repair and sense and respond to their environment. Inspired by this, the field of engineered living materials (ELMs) aims to use genetically-engineered organisms to generate novel materials. Bacterial cellulose (BC) is a biological material with impressive physical properties and low cost of production that is an attractive substrate for ELMs. Inspired by how plants build materials from tissues with specialist cells we here developed a system for making novel BC-based ELMs by addition of engineered yeast programmed to add functional traits to a cellulose matrix. This is achieved via a synthetic ‘symbiotic culture of bacteria and yeast’ (Syn-SCOBY) approach that uses a stable co-culture of <jats:italic>Saccharomyces cerevisiae</jats:italic> with BC-producing <jats:italic>Komagataeibacter rhaeticus</jats:italic> bacetria. Our Syn-SCOBY approach allows inoculation of engineered cells into simple growth media, and under mild conditions materials self-assemble with genetically-programmable functional properties in days. We show that co-cultured yeast can be engineered to secrete enzymes into BC, generating autonomously grown catalytic materials and enabling DNA-encoded modification of BC bulk material properties. We further developed a method for incorporating <jats:italic>S. cerevisiae</jats:italic> within the growing cellulose matrix, creating living materials that can sense chemical and optical inputs. This enabled growth of living sensor materials that can detect and respond to environmental pollutants, as well as living films that grow images based on projected patterns. This novel and robust Syn-SCOBY system empowers the sustainable productio
Gowers G-OF, Cameron SJS, Perdones-Montero A, et al., 2019, Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry, ACS Synthetic Biology, Vol: 8, Pages: 2566-2575, ISSN: 2161-5063
Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) - a form of ambient laser desorption ionisation mass spectrometry - to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.
Gowers G-O, Vince O, Charles J-H, et al., 2019, Entirely off-grid and solar-powered DNA sequencing of microbial communities during an ice cap traverse expedition, Genes, Vol: 10, Pages: 1-10, ISSN: 2073-4425
Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth’s surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions. While remote research stations have significantly lowered the barrier to studying the microbial communities on icecaps, their use has led to a bias for data collection in the near vicinity of these institutions. Here, miniaturisation of a DNA sequencing lab suitable for off-grid metagenomic studies is demonstrated. Using human power alone, this lab was transported across Europe’s largest ice cap (Vatnajökull, Iceland) by ski and sledge. After 11 days of unsupported polar-style travel, a metagenomic study of a geothermal hot spring gorge was conducted on the remote northern edge of the ice cap. This tent-based metagenomic study resulted in over 24 h of Nanopore sequencing, powered by solar power alone. This study demonstrates the ability to conduct DNA sequencing in remote locations, far from civilised resources (mechanised transport, external power supply, internet connection, etc.), whilst greatly reducing the time from sample collection to data acquisition.
Ostrov N, Beal J, Ellis T, et al., 2019, Technological challenges and milestones for writing genomes., Science, Vol: 366, Pages: 310-312, ISSN: 0036-8075
Engineering biology with recombinant DNA, broadly called synthetic biology, has progressed tremendously in the last decade, owing to continued industrialization of DNA synthesis, discovery and development of molecular tools and organisms, and increasingly sophisticated modeling and analytic tools. However, we have yet to understand the full potential of engineering biology because of our inability to write and test whole genomes, which we call synthetic genomics. Substantial improvements are needed to reduce the cost and increase the speed and reliability of genetic tools. Here, we identify emerging technologies and improvements to existing methods that will be needed in four major areas to advance synthetic genomics within the next 10 years: genome design, DNA synthesis, genome editing, and chromosome construction (see table). Similar to other large-scale projects for responsible advancement of innovative technologies, such as the Human Genome Project, an international, cross-disciplinary effort consisting of public and private entities will likely yield maximal return on investment and open new avenues of research and biotechnology.
Walker K, Goosens V, Das A, et al., 2019, Engineered cell-to-cell signalling within growing bacterial cellulose pellicles, Microbial Biotechnology, Vol: 12, Pages: 611-619, ISSN: 1751-7915
Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in health care, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell‐to‐cell communication into a bacterial cellulose‐producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl‐homoserine lactone. We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell‐to‐cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.
Det-Udom R, Gilbert C, Liu L, et al., 2019, Towards semi-synthetic microbial communities: Enhancing soy sauce fermentation properties in B. subtilis co-cultures, Microbial Cell Factories, Vol: 18, ISSN: 1475-2859
BackgroundMany fermented foods and beverages are produced through the action of complex microbial communities. Synthetic biology approaches offer the ability to genetically engineer these communities to improve the properties of these fermented foods. Soy sauce is a fermented condiment with a vast global market. Engineering members of the microbial communities responsible for soy sauce fermentation may therefore lead to the development of improved products. One important property is the colour of soy sauce, with recent evidence pointing to a consumer preference for more lightly-coloured soy sauce products for particular dishes.ResultsHere we show that a bacterial member of the natural soy sauce fermentation microbial community, Bacillus, can be engineered to reduce the ‘browning’ reaction during soy sauce production. We show that two approaches result in ‘de-browning’: engineered consumption of xylose, an important precursor in the browning reaction, and engineered degradation of melanoidins, the major brown pigments in soy sauce. Lastly, we show that these two strategies work synergistically using co-cultures to result in enhanced de-browning.ConclusionsOur results demonstrate the potential of using synthetic biology and metabolic engineering methods for fine-tuning the process of soy sauce fermentation and indeed for many other natural food and beverage fermentations for improved products.
Ellis T, 2019, What is synthetic genomics anyway?, The Biochemist, Vol: 41, Pages: 6-9, ISSN: 0954-982X
You may have heard of synthetic genomics. This headline-grabbing, high-profile, big science topic is starting to emerge catalysed by the pioneering work of famous names in synthetic biology and biotechnology like George Church and Craig Venter. But what is synthetic genomics and what is it being used for? As a prominent researcher at a recent UK meeting said: “Is it just synthetic biology with bigger bits of DNA?” Well no, not quite…
Rajakumar PD, Gower G, Suckling L, et al., 2019, Rapid prototyping platform for Saccharomyces cerevisiae using computer-aided genetic design enabled by parallel software and workcell platform development, Slas Technology, Vol: 24, Pages: 291-297, ISSN: 2472-6303
Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.
Shaw W, Yamauchi H, Mead J, et al., 2019, Engineering a model cell for rational tuning of GPCR signaling, Cell, Vol: 177, Pages: 782-796.e27, ISSN: 0092-8674
G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond tospecific cues in their environment. However, the relationship between stimulus and response for eachGPCR is difficult to predict due to diversity in natural signal transduction architecture and expression.Using genome engineering in yeast, we here constructed an insulated, modular GPCR signaltransduction system to study how the response to stimuli can be predictably tuned using synthetictools. We delineated the contributions of a minimal set of key components via computational andexperimental refactoring, identifying simple design principles for rationally tuning the dose-response.Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineeredto respond to desired concentrations of peptides, metabolites, and hormones relevant to humanhealth. This work enables rational tuning of cell sensing, while providing a framework to guidereprogramming of GPCR-based signaling in other systems.
McCarty NS, Shaw WM, Ellis T, et al., 2019, Rapid assembly of gRNA arrays via modular cloning in yeast, ACS Synthetic Biology, Vol: 8, Pages: 906-910, ISSN: 2161-5063
CRISPR is a versatile technology for genomic editing and regulation, but the expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and expressed from a single transcriptional unit. We demonstrate that these gRNA arrays can stably regulate gene expression in a synergistic manner via dCas9-mediated repression. This approach expands the number of gRNAs that can be expressed in this model organism and may enable the versatile editing or transcriptional regulation of a greater number of genes in vivo.
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.
Stan G, Ellis T, Boo A, 2019, Host-Aware Synthetic Biology, Current Opinion in Systems Biology, ISSN: 2452-3100
Gilbert C, Ellis T, 2019, Biological engineered living materials - growing functional materials with genetically-programmable properties, ACS Synthetic Biology, Vol: 8, Pages: 1-15, ISSN: 2161-5063
Natural biological materials exhibit remarkable properties: self-assembly from simple raw materials, precise control of morphology, diverse physical and chemical properties, self-repair and the ability to sense-and-respond to environmental stimuli. Despite having found numerous uses in human industry and society, the utility of natural biological materials is limited. But, could it be possible to genetically program microbes to create entirely new and useful biological materials? At the intersection between microbiology, material science and synthetic biology, the emerging field of biological Engineered Living Materials (ELMs) aims to answer this question. Here we review recent efforts to program cells to produce living materials with novel functional properties, focussing on microbial systems that can be engineered to grow materials and on new genetic circuits for pattern formation that could be used to produce the more complex systems of the future.
Ellis T, 2019, Predicting how evolution will beat us, MICROBIAL BIOTECHNOLOGY, Vol: 12, Pages: 41-43, ISSN: 1751-7915
Blount B, Ellis T, 2019, The Synthetic Genome Summer Course, Synthetic Biology, Vol: 3, ISSN: 2397-7000
The Synthetic Genome Summer Course was convened with the aim of teaching a wide range of researchers the theory and practical skills behind recent advances in synthetic biology and synthetic genome science, with a focus on Sc2.0, the synthetic yeast genome project. Through software workshops, tutorials and research talks from leading members of the field, the 30 attendees learnt about relevant principles and techniques that they were then able to implement first-hand in laboratory-based practical sessions. Participants SCRaMbLEd semi-synthetic yeast strains to diversify heterologous pathways, used automation to build combinatorial pathway libraries and used CRISPR to debug fitness defects caused by synthetic chromosome design changes. Societal implications of synthetic chromosomes were explored and industrial stakeholders discussed synthetic biology from a commercial standpoint. Over the 5 days, participants gained valuable insight and acquired skills to aid them in future synthetic genome research.
Weenink T, van der Hilst J, McKiernan R, et al., 2019, Design of RNA hairpin modules that predictably tune translation in yeast, Synthetic Biology, Vol: 3, ISSN: 2397-7000
Modular parts for tuning translation are prevalent in prokaryotic synthetic biology but lacking for eukaryotic synthetic biology. Working in Saccharomyces cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5′ untranslated region (5′UTR) of mRNAs can be used to tune expression levels by 100-fold by inhibiting translation. We determine the relationship between the calculated free energy of folding in the 5′UTR and in vivo protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this new tool, computational RNA sequence design can be used to predictably fine-tune protein production for genes expressed in yeast.
Gorochowski TE, Ellis T, 2018, Designing efficient translation, NATURE BIOTECHNOLOGY, Vol: 36, Pages: 934-935, ISSN: 1087-0156
Oling D, Lawenius L, Shaw W, et al., 2018, Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling, ACS SYNTHETIC BIOLOGY, Vol: 7, Pages: 2317-2321, ISSN: 2161-5063
Shaw WM, Yamauchi H, Mead J, et al., 2018, Engineering a model cell for rational tuning of GPCR signaling, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we here constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose-response. Using four different receptors, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites and hormones relevant to human health. This work enables rational tuning of cell sensing, while providing a framework to guide reprogramming of GPCR-based signaling in more complex systems.</jats:p>
Ceroni F, Ellis T, 2018, The challenges facing synthetic biology in eukaryotes, Nature Reviews Molecular Cell Biology, Vol: 19, Pages: 481-482, ISSN: 1471-0072
Synthetic biology is maturing into a true engineering discipline for model microorganisms, but remains far from straightforward for most eukaryotes. Here, we outline the key challenges facing those trying to engineer biology across eukaryota and suggest areas of focus that will aid future progress.
Blount B, Gowers G, Ho JCH, et al., 2018, Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome, Nature Communications, Vol: 9, ISSN: 2041-1723
Synthetic biology tools, such as modular parts and combinatorial DNA assembly, are routinely used to optimise the productivity of heterologous metabolic pathways for biosynthesis or substrate utilisation, yet, it is well established that host strain background is just as important for determining productivity. Here we report that in vivo combinatorial genomic rearrangement of Saccharomyces cerevisiae yeast with a synthetic chromosome V can rapidly generate new, improved host strains with genetic backgrounds favourable to diverse heterologous pathways, including those for violacein and penicillin biosynthesis and for xylose utilisation. We show how the modular rearrangement of synthetic chromosomes by SCRaMbLE can be easily determined using long-read nanopore sequencing and we explore experimental conditions that optimise diversification and screening. This new synthetic genome approach to metabolic engineering provides productivity improvements in a fast, simple and accessible way, making it a valuable addition to existing strain improvement techniques.
Borkowski O, Bricio C, Murgiano M, et al., 2018, Cell-free prediction of protein expression costs for growing cells, Nature Communications, Vol: 9, ISSN: 2041-1723
Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any given gene is a major challenge, as multiple processes and factors combine to determine translation efficiency. To enable prediction of the cost of gene expression in bacteria, we describe here a standard cell-free lysate assay that provides a relative measure of resource consumption when a protein coding sequence is expressed. These lysate measurements can then be used with a computational model of translation to predict the in vivo burden placed on growing E. coli cells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.
Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.
Pothoulakis G, Ellis T, 2018, Construction of hybrid regulated mother-specific yeast promoters for inducible differential gene expression, PLoS ONE, Vol: 13, ISSN: 1932-6203
Engineered promoters with predefined regulation are a key tool for synthetic biology that enable expression on demand and provide the logic for genetic circuits. To expand the availability of synthetic biology tools for S. cerevisiae yeast, we here used hybrid promoter engineering to construct tightly-controlled, externally-inducible promoters that only express in haploid mother cells that have contributed a daughter cell to the population. This is achieved by combining elements from the native HO promoter and from a TetR-repressible synthetic promoter, with the performance of these promoters characterized by both flow cytometry and microfluidics-based fluorescence microscopy. These new engineered promoters are provided as an enabling tool for future synthetic biology applications that seek to exploit differentiation within a yeast population.
Pothoulakis G, Ellis T, 2018, Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype, Communications Biology, Vol: 1, ISSN: 2399-3642
Pseudohyphal growth is a multicellular phenotype naturally performed by wild budding yeast cells in response to stress. Unicellular yeast cells undergo gross changes in their gene regulation and elongate to form branched filament structures consisting of connected cells. Here, we construct synthetic gene regulation systems to enable external induction of pseudohyphal growth in Saccharomyces cerevisiae. By controlling the expression of the natural PHD1 and FLO8 genes we are able to trigger pseudohyphal growth in both diploid and haploid yeast, even in different types of rich media. Using this system, we also investigate how members of the BUD gene family control filamentation in haploid cells. Finally, we employ a synthetic genetic timer network to control pseudohyphal growth and further explore the reversibility of differentiation. Our work demonstrates that synthetic regulation can exert control over a complex multigene phenotype and offers opportunities for rationally modifying the resulting multicellular structure.
Wintle BC, Boehm CR, Rhodes C, et al., 2017, A transatlantic perspective on 20 emerging issues in biological engineering, eLife, Vol: 6, ISSN: 2050-084X
Advances in biological engineering are likely to have substantial impacts on global society. To explorethese potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunitiesand risks presented by biological engineering. We first identified 70 potential issues, and then used an iterativeprocess to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to berelatively unknown outside the field of biological engineering. The issues identified may be of interest toresearchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.
Mitchell LA, Ellis T, 2017, Synthetic genome engineering gets infectious, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 114, Pages: 11006-11008, ISSN: 0027-8424
Jewett MC, Ellis T, 2017, Editorial overview: Synthetic biology: Frontiers in synthetic biology, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 40, Pages: A1-A3, ISSN: 1367-5931
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