Publications
325 results found
Weston DJ, Russell RA, Batty E, et al., 2015, New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts, JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 12, ISSN: 1742-5689
Kelwick R, Kopniczky M, Bower I, et al., 2015, A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered <i>Escherichia coli</i>, PLOS ONE, Vol: 10, ISSN: 1932-6203
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- Citations: 8
Campeotto I, Zhang Y, Mladenov MG, et al., 2015, Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family, Journal of Biological Chemistry, Vol: 290, Pages: 2888-2901, ISSN: 1083-351X
Signaling nucleotides are integral parts of signal transductionsystems allowing bacteria to cope with and rapidly respond tochanges in the environment. The Staphylococcus aureus PII-likesignal transduction protein PstA was recently identified as acyclic diadenylate monophosphate (c-di-AMP)-binding protein.Here, we present the crystal structures of the apo- and c-diAMP-boundPstA protein, which is trimeric in solution as wellas in the crystals. The structures combined with detailed bioinformaticsanalysis revealed that the protein belongs to a newfamily of proteins with a similar core fold but with distinct featuresto classical PII proteins, which usually function in nitrogenmetabolism pathways in bacteria. The complex structurerevealed three identical c-di-AMP-binding sites per trimer witheach binding site at a monomer-monomer interface. Althoughdistinctly different from other cyclic-di-nucleotide-bindingsites, as the half-binding sites are not symmetrical, the complexstructure also highlighted common features for c-di-AMPbindingsites. A comparison between the apo and complexstructures revealed a series of conformational changes thatresult in the ordering of two anti-parallel !-strands that protrudefrom each monomer and allowed us to propose a mechanismon how the PstA protein functions as a signaling transductionprotein.
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.
Kelwick R, MacDonald JT, Webb AJ, et al., 2014, Developments in the Tools and Methodologies of Synthetic Biology, Frontiers in Bioengineering and Biotechnology, Vol: 2
Foerster A, Planamente S, Manoli E, et al., 2014, Coevolution of the ATPase ClpV, the Sheath Proteins TssB and TssC, and the Accessory Protein TagJ/HsiE1 Distinguishes Type VI Secretion Classes, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289
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- Citations: 43
Campeotto I, Percy MG, MacDonald JT, et al., 2014, Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System, Journal of Biological Chemistry, Vol: 289, Pages: 28054-28069, ISSN: 0021-9258
Lipoteichoic acid (LTA) is an important cell wall componentrequired for proper cell growth in many Gram-positive bacteria.In Listeria monocytogenes, two enzymes are required for the synthesisof this polyglycerolphosphate polymer. The LTA primaseLtaPLm initiates LTA synthesis by transferring the first glycerolphosphate(GroP) subunit onto the glycolipid anchor and theLTA synthase LtaSLm extends the polymer by the repeated additionof GroP subunits to the tip of the growing chain. Here, wepresent the crystal structures of the enzymatic domains ofLtaPLm and LtaSLm. Although the enzymes share the same fold,substantial differences in the cavity of the catalytic site andsurface charge distribution contribute to enzyme specialization.The eLtaSLm structure was also determined in complexwith GroP revealing a second GroP binding site. Mutationalanalysis confirmed an essential function for this binding siteand allowed us to propose a model for the binding of thegrowing chain.
Polizzi KM, Kylilis N, Lai HE, et al., 2014, Detecting protein biomarkers using engineered biosensors based on synthetic biology principles, 248th National Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
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
Goers L, Freemont P, Polizzi KM, 2014, Co-culture systems and technologies: taking synthetic biology to the next level, JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 11, ISSN: 1742-5689
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- Citations: 345
Ewens CA, Panico S, Kloppsteck P, et al., 2014, The p97-FAF1 Protein Complex Reveals a Common Mode of p97 Adaptor Binding, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289, Pages: 12077-12084
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- Citations: 20
Yeung HO, Foerster A, Bebeacua C, et al., 2014, Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy, Open Biology, Vol: 4, ISSN: 2046-2441
The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.
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
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.
MacDonald JT, Kelley LA, Freemont PS, 2013, Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling, PLOS One, Vol: 8, ISSN: 1932-6203
Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing the degrees of freedom. The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using -carbon positions only and sampling conformations with a Monte Carlo simulated annealing based protocol. Backbone atoms were added using a method previously described and then gradient minimised in the Rosetta force field. Despite the CG potential energy function being sequence-independent, the method performed similarly to methods that explicitly use either fragments of known protein backbones with similar sequences or residue-specific /-maps to restrict the search space. The method was also able to predict with sub-Angstrom accuracy two out of seven loops from recently solved crystal structures of proteins with low sequence and structure similarity to previously deposited structures in the PDB. The ability to sample realistic loop conformations directly from a potential energy function enables the incorporation of additional geometric restraints and the use of more advanced sampling methods in a way that is not possible to do easily with fragment replacement methods and also enable multi-scale simulations for protein design and protein structure prediction. These restraints could be derived from experimental data or could be design restraints in the case of computational protein design. C++ source code is available for download from http://www.sbg.
Chappell J, Jensen K, Freemont PS, 2013, Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology, Nucleic Acids Research, Vol: 41, Pages: 3471-3481, ISSN: 0305-1048
A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.
Chappell J, Freemont P, 2013, In Vivo and In Vitro Characterization of σ<SUP>70</SUP> Constitutive Promoters by Real-Time PCR and Fluorescent Measurements, SYNTHETI C BIOLOGY, Vol: 1073, Pages: 61-74, ISSN: 1064-3745
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- Citations: 3
Goers L, Kylilis N, Tomazou M, et al., 2013, Engineering Microbial Biosensors, MICROBIAL SYNTHETIC BIOLOGY, Editors: Harwood, Wipat, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 119-156, ISBN: 978-0-12-417029-2
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- Citations: 11
Boehm CR, Freemont PS, Ces O, 2013, Design of a prototype flow microreactor for synthetic biology <i>in vitro</i>, LAB ON A CHIP, Vol: 13, Pages: 3426-3432, ISSN: 1473-0197
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- Citations: 25
Odendall C, Rolhion N, Foerster A, et al., 2012, The <i>Salmonella</i> Kinase SteC Targets the MAP Kinase MEK to Regulate the Host Actin Cytoskeleton, CELL HOST & MICROBE, Vol: 12, Pages: 657-668, ISSN: 1931-3128
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- Citations: 60
Lu D, Silhan J, MacDonald JT, et al., 2012, Structural basis for the recognition and cleavage of abasic DNA in <i>Neisseria meningitidis</i>, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 16852-16857, ISSN: 0027-8424
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- Citations: 16
Lossi NS, Manoli E, Simpson P, et al., 2012, The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system, MOLECULAR MICROBIOLOGY, Vol: 86, Pages: 437-456, ISSN: 0950-382X
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- Citations: 20
Kitney R, Freemont P, 2012, Synthetic biology - the state of play, FEBS LETTERS, Vol: 586, Pages: 2029-2036, ISSN: 0014-5793
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- Citations: 51
Weston DJ, Adams NM, Russell RA, et al., 2012, Analysis of spatial point patterns in nuclear biology, PLoS ONE, Vol: 7, ISSN: 1932-6203
There is considerable interest in cell biology in determining whether, and to what extent, the spatial arrangement of nuclear objects affects nuclear function. A common approach to address this issue involves analyzing a collection of images produced using some form of fluorescence microscopy. We assume that these images have been successfully pre-processed and a spatial point pattern representation of the objects of interest within the nuclear boundary is available. Typically in these scenarios, the number of objects per nucleus is low, which has consequences on the ability of standard analysis procedures to demonstrate the existence of spatial preference in the pattern. There are broadly two common approaches to look for structure in these spatial point patterns. First a spatial point pattern for each image is analyzed individually, or second a simple normalization is performed and the patterns are aggregated. In this paper we demonstrate using synthetic spatial point patterns drawn from predefined point processes how difficult it is to distinguish a pattern from complete spatial randomness using these techniques and hence how easy it is to miss interesting spatial preferences in the arrangement of nuclear objects. The impact of this problem is also illustrated on data related to the configuration of PML nuclear bodies in mammalian fibroblast cells.
Niwa H, Ewens CA, Tsang C, et al., 2012, The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 8561-8570
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- Citations: 90
Silhan J, Nagorska K, Zhao Q, et al., 2012, Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis, Nucleic Acids Research, Vol: 40, Pages: 2065-2075, ISSN: 1362-4962
We have previously demonstrated that the twoExonuclease III (Xth) family members presentwithin the obligate human pathogen Neisseriameningitidis, NApe and NExo, are important forsurvival under conditions of oxidative stress. Ofthese, only NApe possesses AP endonucleaseactivity, while the primary function of NExoremained unclear. We now reveal further functionalspecialization at the level of 30-PO4 processing forNExo. We demonstrate that the bi-functional meningococcalglycosylases Nth and MutM can performstrand incisions at abasic sites in addition to NApe.However, no such functional redundancy existsfor the 30-phosphatase activity of NExo, and thecytotoxicity of 30-blocking lesions is reflectedin the marked sensitivity of a mutant lackingNExo to oxidative stress, compared to strainsdeficient in other base excision repair enzymes. Ahistidine residue within NExo that is responsiblefor its lack of AP endonuclease activity isalso important for its 30-phosphatase activity,demonstrating an evolutionary trade off in enzymefunction at the single amino acid level. This specializationof two Xth enzymes for the 30-end processingand strand-incision reactions has notpreviously been observed and provides a newparadigm within the prokaryotic world for separationof these critical functions during baseexcision repair.
Nagorska K, Silhan J, Li Y, et al., 2012, A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis, MOLECULAR MICROBIOLOGY, Vol: 83, Pages: 1064-1079, ISSN: 0950-382X
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- Citations: 20
Bebeacua C, Förster A, McKeown C, et al., 2012, Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy., Proc Natl Acad Sci U S A, Vol: 109, Pages: 1098-1103
p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.
Kloppsteck P, Ewens CA, Foerster A, et al., 2012, Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, Vol: 1823, Pages: 125-129, ISSN: 0167-4889
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- Citations: 55
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