26 results found
Kopniczky MB, Canavan C, McClymont DW, et al., 2020, Cell-free protein synthesis as a prototyping platform for mammalian synthetic biology, ACS Synthetic Biology, Vol: 9, Pages: 144-156, ISSN: 2161-5063
The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.
Appuswamy R, Brigand KL, Barbry P, et al., 2019, OligoArchive: Using DNA in the DBMS storage hierarchy, CIDR 2019
Freemont PS, Moore S, MacDonald J, et al., 2018, Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from non-model bacteria, Proceedings of the National Academy of Sciences, Vol: 115, Pages: E4340-E4349, ISSN: 0027-8424
Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.
Freemont PS, Salih O, He S, et al., 2018, Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa, Structure, Vol: 26, Pages: 329-336.e3, ISSN: 0969-2126
Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.
MacDonald JT, Siciliano V, 2017, Computational Sequence Design with R2oDNA Designer., Methods Mol Biol, Vol: 1651, Pages: 249-262
Recently developed DNA assembly methods have enabled the rapid and simultaneous assembly of multiple parts to create complex synthetic gene circuits. A number of groups have proposed the use of computationally designed orthogonal spacer sequences to guide the ordered assembly of parts using overlap-directed or homologous recombination-based methods. This approach is particularly useful for assembling multiple parts with repetitive elements. Orthogonal spacer sequences (sometimes called UNSs-unique nucleotide sequences) also have a number of other potential uses including in the design of synthetic promoters regulated by novel regulatory elements.
Moore SJ, macdonald JT, freemont PS, 2017, Cell-free synthetic biology for in vitro prototype engineering, Biochemical Society Transactions, Vol: 45, Pages: 785-791, ISSN: 1470-8752
Cell-free transcription–translation is an expandingfield in synthetic biology as a rapidprototyping platform for blueprinting the design of synthetic biological devices. Exemplarefforts include translation of prototype designs into medical test kits for on-site identifica-tion of viruses (Zika and Ebola), while gene circuit cascades can be tested, debuggedand re-designed within rapid turnover times. Coupled with mathematical modelling, thisdiscipline lends itself towards the precision engineering of new synthetic life. The nextstages of cell-free look set to unlock new microbial hosts that remain slow to engineerand unsuited to rapid iterative design cycles. It is hoped that the development of suchsystems will provide new tools to aid the transition from cell-free prototype designs tofunctioning synthetic genetic circuits and engineered natural product pathways in livingcells.
MacDonald J, Freemont PS, 2016, Computational protein design with backbone plasticity, Biochemical Society Transactions, Vol: 44, Pages: 1523-1529, ISSN: 1470-8752
The computational algorithms used in the design of artificial proteins have become increasingly sophisticated in recent years, producing a series of remarkable successes. The most dramatic of these is the de novo design of artificial enzymes. The majority of these designs have reused naturally occurring protein structures as ‘scaffolds’ onto which novel functionality can be grafted without having to redesign the backbone structure. The incorporation of backbone flexibility into protein design is a much more computationally challenging problem due to the greatly increased search space, but promises to remove the limitations of reusing natural protein scaffolds. In this review, we outline the principles of computational protein design methods and discuss recent efforts to consider backbone plasticity in the design process.
Kelwick RJR, Webb AJ, MacDonald JT, et al., 2016, Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements, Metabolic Engineering, Vol: 38, Pages: 370-381, ISSN: 1096-7184
Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.
MacDonald JT, Kabasakal BV, Godding D, et al., 2016, Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 10346-10351, ISSN: 1091-6490
The ability to design and construct structures with atomic level precisionis one of the key goals of nanotechnology. Proteins offer anattractive target for atomic design, as they can be synthesized chemicallyor biologically, and can self-assemble. However the generalizedprotein folding and design problem is unsolved. One approach tosimplifying the problem is to use a repetitive protein as a scaffold.Repeat proteins are intrinsically modular, and their folding and structuresare better understood than large globular domains. Here, wehave developed a new class of synthetic repeat protein, based onthe pentapeptide repeat family of beta-solenoid proteins. We haveconstructed length variants of the basic scaffold, and computationallydesigned de novo loops projecting from the scaffold core. Theexperimentally solved 3.56 ˚A resolution crystal structure of one designedloop matches closely the designed hairpin structure, showingthe computational design of a backbone extension onto a syntheticprotein core without the use of backbone fragments from knownstructures. Two other loop designs were not clearly resolved in thecrystal structures and one loop appeared to be in an incorrect conformation.We have also shown that the repeat unit can accommodatewhole domain insertions by inserting a domain into one of the designedloops.
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.
Yu N, Nützmann HW, MacDonald JT, et al., 2016, Delineation of metabolic gene clusters in plant genomes by chromatin signatures., Nucleic Acids Research, Vol: 44, Pages: 2255-2265, ISSN: 1362-4962
Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.
Kelwick R, Webb AJ, Macdonald JT, et al., 2016, Development of a bacillus subtilis cell-free transcriptiontranslation system
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
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.
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
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.
Moore BL, Kelley LA, Barber J, et al., 2013, High-quality protein backbone reconstruction from alpha carbons using gaussian mixture models, JOURNAL OF COMPUTATIONAL CHEMISTRY, Vol: 34, Pages: 1881-1889, ISSN: 0192-8651
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.
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
Coluzza I, MacDonald JT, Sadowski MI, et al., 2012, Analytic Markovian Rates for Generalized Protein Structure Evolution, PLOS ONE, Vol: 7, ISSN: 1932-6203
Sadowski MI, MacDonald JT, 2011, Designing New Proteins, Amino Acids, Peptides and Proteins in Organic Chemistry, Protection Reactions, Medicinal Chemistry, Combinatorial Synthesis, Editors: Hughes, Publisher: Wiley-VCH, ISBN: 9783527321032
Proteins provide living organisms with a flexible tool kit for highly specific catalysis, binding, transport, and structural properties by combination of the 20 amino acids into linear polypeptides and the assembly of polypeptides into multisubunit complexes. Once the rules governing folding, stability, and chemistry are understood new proteins can be engineered for applications in materials science, nanotechnology, biochemical engineering, and medicine. The past two decades have seen several significant advances in the creation of new proteins by computational design and reengineering by directed evolution. In this chapter, we provide an overview of the most important current methods in computational and laboratory-based protein design, describe significant successes in the design of protein structures, functions, and interactions, and discuss important future goals for further development.
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
Macdonald JT, Purkiss AG, Smith MA, et al., 2005, Unfolding crystallins: The destabilizing role of a beta-hairpin cysteine in beta B2-crystallin by simulation and experiment, PROTEIN SCIENCE, Vol: 14, Pages: 1282-1292, ISSN: 0961-8368
Purkiss AG, Macdonald JT, Goodfellow JM, et al., 2004, Comparison of generalised born/surface area with periodic boundary simulations to study protein unfolding, MOLECULAR SIMULATION, Vol: 30, Pages: 333-340, ISSN: 0892-7022
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