Publications
106 results found
Storch M, Casini A, Mackrow B, et al., 2016, BASIC: A Simple and Accurate Modular DNA Assembly Method., Methods Mol Biol, Vol: 1472, Pages: 79-91
Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts [1]. The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round [2].
Awan A, Blount B, Bell D, et al., 2016, Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast, Biorxiv
Abstract Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. By taking genes from a filamentous fungus and directing their efficient expression and subcellular localisation, we here engineer the baker’s yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide. Using synthetic biology tools combined with long-read DNA sequencing, we optimise productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.
Ceroni F, Blount BA, Ellis T, 2016, Sensing the Right Time to Be Productive, Cell Systems, Vol: 3, Pages: 116-117, ISSN: 2405-4720
Engineered E. coli can be made to autonomously switch from growth to production by a modular two-gate system that reduces the burden of biosynthesis.
Borkowski O, Ceroni F, Stan GB, et al., 2016, Overloaded and stressed: whole-cell considerations for bacterial synthetic biology., Current Opinion in Microbiology, Vol: 33, Pages: 123-130, ISSN: 1879-0364
The predictability and robustness of engineered bacteria depend on the many interactions between synthetic constructs and their host cells. Expression from synthetic constructs is an unnatural load for the host that typically reduces growth, triggers stresses and leads to decrease in performance or failure of engineered cells. Work in systems and synthetic biology has now begun to address this through new tools, methods and strategies that characterise and exploit host-construct interactions in bacteria. Focusing on work in E. coli, we review here a selection of the recent developments in this area, highlighting the emerging issues and describing the new solutions that are now making the synthetic biology community consider the cell just as much as they consider the construct.
Borkowski O, Gilbert C, Ellis T, 2016, SYNTHETIC BIOLOGY. On the record with E. coli DNA., Science, Vol: 353, Pages: 444-445, ISSN: 0036-8075
Reeve B, Martinez Klimova E, de Jonghe J, et al., 2016, The Geobacillus plasmid set: a modular toolkit for thermophile engineering, ACS Synthetic Biology, Vol: 5, Pages: 1342-1347, ISSN: 2161-5063
Geobacillus thermoglucosidasius is agram-positive thermophile of industrial interest thatexhibits rapid growth and can utilize a variety ofplant-derived feedstocks. It is an attractive chassisorganism for high temperature biotechnology andsynthetic biology applications but is currently limitedby a lack of available genetic tools. Here we describea set of modular shuttle vectors, including apromoter library and reporter proteins. The compactplasmids are composed of interchangeable modulesfor molecular cloning in Escherichia coli and stablepropagation in G. thermoglucosidasius and otherGeobacillus species. Modules include two origins ofreplication, two selectable markers and three reporterproteins for characterization of gene expression.For fine-tuning heterologous expression from theseplasmids, we include a characterized promoter libraryand test ribosome binding site design. Together,these gene expression tools and a standardizedplasmid set can facilitate modularity and part exchangeto make Geobacillus a thermophile chassis forsynthetic biology.
Florea M, Hagemann H, Santosa G, et al., 2016, Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E3431-E3440, ISSN: 0027-8424
Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity and biocompatibility make it of great interest to materials science, however precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a new strain of Komagataeibacter rhaeticus (Komagataeibacter rhaeticus iGEM) that can produce cellulose at high yields, grow in low nitrogen conditions, and is highly resistant to toxic chemicals. We achieve external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering and biotechnology.
Blount BA, Driessen MRM, Ellis T, 2016, GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA, Scientific Reports, Vol: 6, ISSN: 2045-2322
Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb.
Webb AJ, Kelwick R, Doenhoff MJ, et al., 2016, A protease-based biosensor for the detection of schistosome cercariae, Scientific Reports, Vol: 6, ISSN: 2045-2322
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses anddeath. Rapid and cost-effective approaches to detect parasites are needed, especially inresource-limited settings. A common signature of parasitic diseases is the release of specificproteases by the parasites at multiple stages during their life cycles. To this end, weengineered several modular Escherichia coli and Bacillus subtilis whole-cell-basedbiosensors which incorporate an interchangeable protease recognition motif into theirdesigns. Herein, we describe how several of our engineered biosensors have been applied todetect the presence and activity of elastase, an enzyme released by the cercarial larvae stageof Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes areresponsible for the infection of an estimated 200 million people worldwide. Since ourbiosensors are maintained in lyophilised cells, they could be applied for the detection of S.mansoni and other parasites in settings without reliable cold chain access.
Awan AR, Shaw WM, Ellis T, 2016, Biosynthesis of therapeutic natural products using synthetic biology, Advanced Drug Delivery Reviews, Vol: 105, Pages: 96-106, ISSN: 1872-8294
Natural products are a group of bioactive structurally diverse chemicals produced by microorganisms and plants. These molecules and their derivatives have contributed to over a third of the therapeutic drugs produced in the last century. However, over the last few decades traditional drug discovery pipelines from natural products have become far less productive and far more expensive. One recent development with promise to combat this trend is the application of synthetic biology to therapeutic natural product biosynthesis. Synthetic biology is a young discipline with roots in systems biology, genetic engineering, and metabolic engineering. In this review, we discuss the use of synthetic biology to engineer improved yields of existing therapeutic natural products. We further describe the use of synthetic biology to combine and express natural product biosynthetic genes in unprecedented ways, and how this holds promise for opening up completely new avenues for drug discovery and production.
Florea M, Reeve B, Abbott J, et al., 2016, Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582., Scientific Reports, Vol: 6, ISSN: 2045-2322
Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.
Reeve AB, Petkiewicz S, Hagemann H, et al., 2016, Modified bacterial nanocellulose as a bioadsorbent material
Storch M, Casini A, Mackrow B, et al., 2015, BASIC: a new Biopart Assembly Standard for Idempotent Cloning provides accurate, single-tier DNA assembly for synthetic biology, ACS Synthetic Biology, Vol: 4, Pages: 781-787, ISSN: 2161-5063
The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organisation, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels.
Casini A, Storch M, Baldwin GS, et al., 2015, Bricks and blueprints: methods and standards for DNA assembly, Nature Reviews Molecular Cell Biology, Vol: 16, Pages: 568-576, ISSN: 1471-0080
DNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade, a plethora of powerful new DNA assembly methods — including Gibson Assembly, Golden Gate and ligase cycling reaction (LCR) — have been developed. In this Innovation article, we discuss these methods as well as standards such as the modular cloning (MoClo) system, GoldenBraid, modular overlap-directed assembly with linkers (MODAL) and PaperClip, which have been developed to facilitate a streamlined assembly workflow, to aid the exchange of material between research groups and to create modular reusable DNA parts.
Ceroni F, Algar R, Stan G-B, et al., 2015, Quantifying cellular capacity identifies gene expression designs with reduced burden, Nature Methods, Vol: 12, Pages: 415-418, ISSN: 1548-7105
Heterologous gene expression can be a significant burden forcells. Here we describe an in vivo monitor that tracks changesin the capacity of Escherichia coli in real time and can be usedto assay the burden imposed by synthetic constructs and theirparts. We identify construct designs with reduced burden thatpredictably outperformed less efficient designs, despite havingequivalent output.
Wright O, Delmans M, Stan G-B, et al., 2015, Gene Guard: A Modular Plasnnid System Designed for Biosafety, ACS SYNTHETIC BIOLOGY, Vol: 4, Pages: 307-316, ISSN: 2161-5063
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- Citations: 74
Bricio C, Liu X, Li X, et al., 2015, High-throughput picodroplet-based analysis of biosynthetic libraries, Pages: 709-710
Baldwin G, Bayer T, Dickinson R, et al., 2015, Synthetic biology - a primer, ISBN: 9781783268801
Synthetic Biology - A Primer (Revised Edition) presents an updated overview of the field of synthetic biology and the foundational concepts on which it is built. This revised edition includes new literature references, working and updated URL links, plus some new figures and text where progress in the field has been made. The book introduces readers to fundamental concepts in molecular biology and engineering and then explores the two major themes for synthetic biology, namely ‘bottom-up’ and ‘top-down’ engineering approaches. ‘Top-down’ engineering uses a conceptual framework of systematic design and engineering principles focused around the Design-Build-Test cycle and mathematical modelling. The ‘bottom-up’ approach involves the design and building of synthetic protocells using basic chemical and biochemical building blocks from scratch exploring the fundamental basis of living systems. Examples of cutting-edge applications designed using synthetic biology principles are presented, including: the production of novel, microbial synthesis of pharmaceuticals and fine chemicals the design and implementation of biosensors to detect infections and environmental waste. The book also describes the Internationally Genetically Engineered Machine (iGEM) competition, which brings together students and young researchers from around the world to carry out summer projects in synthetic biology. Finally, the primer includes a chapter on the ethical, legal and societal issues surrounding synthetic biology, illustrating the integration of social sciences into synthetic biology research. Final year undergraduates, postgraduates and established researchers interested in learning about the interdisciplinary field of synthetic biology will benefit from this up-to-date primer on synthetic biology.
Lu M, Williamson N, Boschetti C, et al., 2015, Expression-level dependent perturbation of cell proteostasis and nuclear morphology by aggregation-prone polyglutamine proteins, Biotechnol. Bioeng., Pages: n/a-n/a, ISSN: 1097-0290
We describe a gene expression system for use in mammalian cells that yields reproducible, inducible gene expression that can be modulated within the physiological range. A synthetic promoter library was generated from which representatives were selected that gave weak, intermediate-strength or strong promoter activity. Each promoter resulted in a tight expression range when used to drive single-copy reporter genes integrated at the same genome location in stable cell lines, in contrast to the broad range of expression typical of transiently transfected cells. To test this new expression system in neurodegenerative disease models, we used each promoter type to generate cell lines carrying single-copy genes encoding polyglutamine-containing proteins. Expression over a period of up to three months resulted in a proportion of cells developing juxtanuclear aggresomes whose rate of formation, penetrance and morphology were expression-level dependent. At the highest expression levels, fibrillar aggregates deposit close to the nuclear envelope, indicating that cell proteostasis is overwhelmed by misfolded protein species. We also observed expression-level dependent, abnormal nuclear morphology in cells containing aggresomes, with up to ∼80% of cells affected. This system constitutes a valuable tool in gene regulation at different levels and allows the quantitative assessment of gene expression effects when developing disease models or investigating cell function through the introduction of gene constructs. This article is protected by copyright. All rights reserved
Pothoulakis G, Ellis T, 2015, Using Spinach Aptamer to Correlate mRNA and Protein Levels in <i>Escherichia coli</i>, RIBOSWITCHES AS TARGETS AND TOOLS, Vol: 550, Pages: 173-185, ISSN: 0076-6879
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- Citations: 3
Jovicevic D, Blount BA, Ellis T, 2014, Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast, BIOESSAYS, Vol: 36, Pages: 855-860, ISSN: 0265-9247
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- Citations: 23
Casini A, Christodoulou G, Freemont PS, et al., 2014, R2oDNA Designer: Computational Design of Biologically Neutral Synthetic DNA Sequences, ACS SYNTHETIC BIOLOGY, Vol: 3, Pages: 525-528, ISSN: 2161-5063
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- Citations: 47
Pothoulakis G, Ceroni F, Reeve B, et al., 2014, The spinach RNA aptamer as a characterization tool for synthetic biology, ACS Synthetic Biology, Vol: 3, Pages: 182-187, ISSN: 2161-5063
Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.
Cai Y, Ellis T, 2014, The Sixth International Meeting on synthetic Biology (SB6.0) Special Issue Editorial, ACS SYNTHETIC BIOLOGY, Vol: 3, Pages: 107-107, ISSN: 2161-5063
Reeve B, Hargest T, Gilbert C, et al., 2014, Predicting translation initiation rates for designing synthetic biology., Frontiers in Bioengineering and Biotechnology, Vol: 2, Pages: 1-1, ISSN: 2296-4185
In synthetic biology, precise control over protein expression is required in order to construct functional biological systems. A core principle of the synthetic biology approach is a model-guided design and based on the biological understanding of the process, models of prokaryotic protein production have been described. Translation initiation rate is a rate-limiting step in protein production from mRNA and is dependent on the sequence of the 5'-untranslated region and the start of the coding sequence. Translation rate calculators are programs that estimate protein translation rates based on the sequence of these regions of an mRNA, and as protein expression is proportional to the rate of translation initiation, such calculators have been shown to give good approximations of protein expression levels. In this review, three currently available translation rate calculators developed for synthetic biology are considered, with limitations and possible future progress discussed.
Reeve B, Sanderson T, Ellis T, et al., 2014, How Synthetic Biology Will Reconsider Natural Bioluminescence and Its Applications, BIOLUMINESCENCE: FUNDAMENTALS AND APPLICATIONS IN BIOTECHNOLOGY, VOL 2, Vol: 145, Pages: 3-30, ISSN: 0724-6145
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- Citations: 10
Algar RJR, Ellis T, Stan G-B, 2014, Modelling essential interactions between synthetic genes and their chassis cell, 53rd IEEE Annual Conference on Decision and Control (CDC), Publisher: IEEE, Pages: 5437-5444, ISSN: 0743-1546
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- Citations: 12
Casini A, MacDonald JT, De Jonghe J, et al., 2013, One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy, Nucleic Acids Research, Vol: 42, ISSN: 1362-4962
Overlap-directed DNA assembly methods allowmultiple DNA parts to be assembled together inone reaction. These methods, which rely onsequence homology between the ends of DNAparts, have become widely adopted in syntheticbiology, despite being incompatible with a key principleof engineering: modularity. To answer this, wepresent MODAL: a Modular Overlap-DirectedAssembly with Linkers strategy that brings modularityto overlap-directed methods, allowing assemblyof an initial set of DNA parts into a variety ofarrangements in one-pot reactions. MODAL isaccompanied by a custom software tool thatdesigns overlap linkers to guide assembly,allowing parts to be assembled in any specifiedorder and orientation. The in silico design of syntheticorthogonal overlapping junctions allows formuch greater efficiency in DNA assembly for avariety of different methods compared with usingnon-designed sequence. In tests with three differentassembly technologies, the MODAL strategy givesassembly of both yeast and bacterial plasmids,composed of up to five DNA parts in the kilobaserange with efficiencies of between 75 and 100%.It also seamlessly allows mutagenesis to beperformed on any specified DNA parts duringthe process, allowing the one-step creation of constructlibraries valuable for synthetic biologyapplications.
Algar R, Ellis T, Stan G-B, 2013, Modelling the burden caused by gene expression: an in silico investigation into the interactions between synthetic gene circuits and their chassis cell
In this paper we motivate and develop a model of gene expression for the purpose of studying the interaction between synthetic gene circuits and the chassis cell within which they are inserted. This model focuses on the translational aspect of gene expression as this is where the literature suggests the crucial interaction between gene expression and shared resources lies.
Brucoli F, Hawkins RM, James CH, et al., 2013, An Extended Pyrrolobenzodiazepine-Polyamide Conjugate with Selectivity for a DNA Sequence Containing the ICB2 Transcription Factor Binding Site, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 56, Pages: 6339-6351, ISSN: 0022-2623
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- Citations: 25
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