255 results found
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
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
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
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.
Lossi NS, Manoli E, Foerster A, et al., 2013, The HsiB1C1 (TssB-TssC) Complex of the Pseudomonas aeruginosa Type VI Secretion System Forms a Bacteriophage Tail Sheathlike Structure, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 7536-7548
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.
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
Boehm CR, Freemont PS, Ces O, 2013, Design of a prototype flow microreactor for synthetic biology in vitro, LAB ON A CHIP, Vol: 13, Pages: 3426-3432, ISSN: 1473-0197
Chappell J, Freemont P, 2013, In vivo and in vitro characterization of σ70 constitutive promoters by real-time PCR and fluorescent measurements., Methods Mol Biol, Vol: 1073, Pages: 61-74
The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.
Odendall C, Rolhion N, Foerster A, et al., 2012, The Salmonella Kinase SteC Targets the MAP Kinase MEK to Regulate the Host Actin Cytoskeleton, CELL HOST & MICROBE, Vol: 12, Pages: 657-668, ISSN: 1931-3128
Lu D, Silhan J, MacDonald JT, et al., 2012, Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 16852-16857, ISSN: 0027-8424
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
Kitney R, Freemont P, 2012, Synthetic biology - the state of play, FEBS LETTERS, Vol: 586, Pages: 2029-2036, ISSN: 0014-5793
Kitney RI, 2012, Synthetic Biology - A Primer, Publisher: Imperial College Press London
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
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
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
Batty EC, Jensen K, Freemont PS, 2012, PML NUCLEAR BODIES AND OTHER TRIM-DEFINED SUBCELLULAR COMPARTMENTS, TRIM/RBCC PROTEINS, Vol: 770, Pages: 39-58, ISSN: 0065-2598
Lossi NS, Dajani R, Freemont P, et al., 2011, Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa, MICROBIOLOGY-SGM, Vol: 157, Pages: 3292-3305, ISSN: 1350-0872
Attia M, Foerster A, Rachez C, et al., 2011, Interaction between Nucleosome Assembly Protein 1-like Family Members, JOURNAL OF MOLECULAR BIOLOGY, Vol: 407, Pages: 647-660, ISSN: 0022-2836
MacDonald JT, Barnes C, Kitney RI, et al., 2011, Computational design approaches and tools for synthetic biology, INTEGRATIVE BIOLOGY, Vol: 3, Pages: 97-108, ISSN: 1757-9694
Russell RA, Adams NM, Stephens D, et al., 2011, Methodology for quantitative analysis of 3-D nuclear architecture, Advances in Nuclear Architecture, Pages: 173-187, ISBN: 9789048198986
© Springer Science+Business Media B.V. 2011. All rights reserved. In the past 20 years cell biologists have studied the cell nucleus extensively, aided by advances in cell imaging technology and microscopy. Consequently, the volume of image data of the cell nucleus - and the compartments it contains - is growing rapidly. The spatial organisation of these nuclear compartments is thought to be fundamentally associated with nuclear function. However, the rules that oversee nuclear architecture remain unclear and controversial. As a result, there is an increasing need to replace qualitative visual assessment of microscope images with quantitative and automated methods. Such tools can substantially reduce manual labour and more importantly remove subjective bias. Quantitative methods can also increase the accuracy, sensitivity and reproducibility of data analysis. In this paper, we describe image processing and analysis methodology for the investigation of nuclear architecture, and the application of these methods to quantitatively explore the promyelocytic leukaemia (PML) nuclear bodies (NBs). PML NBs are linked with numerous nuclear functions including transcription and protein degradation. However, we know very little about the three-dimensional (3-D) architecture of PML NBs in relation to each other or within the general volume of the nucleus. Finally, we emphasise methods for the analysis of replicate images (of a given nuclear compartment across different cell nuclei) in order to aggregate information about nuclear architecture.
Adams NM, Freemont PS, 2010, Advances in Nuclear Architecture, Publisher: Springer Verlag, ISBN: 9789048198986
Akiyoshi T, Heathcote DA, Azzi J, et al., 2010, The Role of SPI6 in the Survival of Allograft Derived Dendritic Cells and Tolerance., AMERICAN JOURNAL OF TRANSPLANTATION, Vol: 10, Pages: 561-561, ISSN: 1600-6135
Ewens CA, Kloppsteck P, Foerster A, et al., 2010, Structural and functional implications of phosphorylation and acetylation in the regulation of the AAA+ protein p97, BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE, Vol: 88, Pages: 41-48, ISSN: 0829-8211
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