11 results found
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.
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.
Moore SJ, Lai HE, Needham H, et al., 2017, Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool, Biotechnology Journal, Vol: 12, ISSN: 1860-7314
Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.
Moore SJ, Sowa ST, Schuchardt C, et al., 2017, Elucidation of the biosynthesis of the methane catalyst coenzyme F430, Nature, Vol: 543, Pages: 78-82, ISSN: 0028-0836
Moore SJ, lai H-E, Kelwick R, et al., 2016, EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology, ACS Synthetic Biology, Vol: 5, Pages: 1059-1069, ISSN: 2161-5063
Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology.
Kopniczky M, moore S, freemont P, 2015, Multilevel regulation and translational switches in synthetic biology, IEEE Transactions on Biomedical Circuits and Systems, Vol: 9, Pages: 485-496, ISSN: 1940-9990
In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules. This is about to change as the repertoire of foundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and RNA responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications.
Moore SJ, Mayer MJ, Biedendieck R, et al., 2014, Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements, New Biotechnology, Vol: 31, Pages: 553-561, ISSN: 1871-6784
Deery E, Frank S, Lawrence A, et al., 2014, Synthetic Biology in Metabolic Engineering: From Complex Biochemical Pathways to Compartmentalized Metabolic Processes – a Vitamin Connection, Book Title Reviews in Cell Biology and Molecular MedicineReviews in Cell Biology and Molecular Medicine
Vitamin B12 (cobalamin) is a remarkable nutrient not only because of its structural complexity but also because it is only synthesized by certain bacteria. In order to understand its biosynthesis and to enhance its production, metabolic engineering and synthetic biology strategies have been applied to elucidate this metabolic process; the results of which have shown that intermediates in the pathway are passed from one enzyme to the next by substrate channeling. Knowledge of the pathway is also being used in the design of vitamin analogs that have potential as drug-delivery vehicles. Once its synthesis is complete, cobalamin is required as either a coenzyme or cofactor in a number of different metabolic processes. Some cobalamin-dependent enzymes are found encased within bacterial microcompartments, proteinaceous organelles that house a specific metabolic pathway. The potential to develop these supra-macromolecular structures into bespoke bioreactors by replacing the embedded pathway is discussed.
Moore SJ, Lawrence AD, Biedendieck R, et al., 2013, Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B <sub>12</sub> ), Proceedings of the National Academy of Sciences, Vol: 110, Pages: 14906-14911, ISSN: 0027-8424
<jats:p> It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B <jats:sub>12</jats:sub> , adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in <jats:italic>Bacillus megaterium</jats:italic> and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the d <jats:sub>yz</jats:sub> orbital, rather than the typical d <jats:sub>z</jats:sub> 2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification. </jats:p>
Moore SJ, Biedendieck R, Lawrence AD, et al., 2013, Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway, Journal of Biological Chemistry, Vol: 288, Pages: 297-305, ISSN: 0021-9258
Moore SJ, Warren MJ, 2012, The anaerobic biosynthesis of vitamin B12, Biochemical Society Transactions, Vol: 40, Pages: 581-586, ISSN: 0300-5127
<jats:p>Vitamin B12 (cobalamin) is a cobalt-containing modified tetrapyrrole that is an essential nutrient for higher animals. Its biosynthesis is restricted to certain bacteria and requires approximately 30 enzymatic steps for its complete de novo construction. Remarkably, two distinct biosynthetic pathways exist, which are termed the aerobic and anaerobic routes. The anaerobic pathway has yet to be fully characterized due to the inherent instability of its oxygen-sensitive intermediates. Bacillus megaterium, a bacterium previously used for the commercial production of cobalamin, has a complete anaerobic pathway and this organism is now being used to investigate the anaerobic B12 pathway through the application of recent advances in recombinant protein production. The present paper provides a summary of recent findings in the anaerobic pathway and future perspectives.</jats:p>
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