243 results found
Kelwick RJR, Ricci L, Chee SM, et al., 2019, Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics, Synthetic Biology, Vol: 3, ISSN: 2397-7000
The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimise several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimising PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customised our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimise in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ∼50%. In cell-free transcription-translation prototyping reactions, GC-MS quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ±0.39 µM), C104 ΔPhaC_C319A (4.62 ±1.31 µM) and C101 ΔPhaC_C319A (2.65 ±1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription-translation reactions produced greater yields of 3HB (32.87 ±6.58 µM) and these reactions were also used to characterise a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimising PHAs production.
Kylilis N, Riangrungroj P, Lai H-E, et al., 2019, Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications., ACS Sens
Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine.
Exley K, Reynolds CR, Suckling L, et al., 2019, Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway, JOURNAL OF BIOLOGICAL ENGINEERING, Vol: 13, ISSN: 1754-1611
Tosi T, Hoshiga F, Millership C, et al., 2019, Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM, PLOS PATHOGENS, Vol: 15, ISSN: 1553-7366
Silhan J, Zhao Q, Boura E, et al., 2018, Structural basis for recognition and repair of the 3 '-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis, NUCLEIC ACIDS RESEARCH, Vol: 46, Pages: 11980-11989, ISSN: 0305-1048
Webb AJ, Allan F, Kelwick R, et al., 2018, Protease-based bioreporters for the detection of schistosome cercariae, American Society of Tropical Medicine and Hygiene (ASTMH) 67th Annual Meeting, New Orleans, Louisiana, USA
Kylilis N, Riangrungroj P, Lai H-E, et al., 2018, A low-cost biological agglutination assay for medical diagnostic applications
Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.
Rajakumar PD, Gowers G-OF, Suckling L, et al., 2018, Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development., SLAS Technol
Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.
Thaore V, Moore S, Polizzi K, et al., Techno-economic evaluation of a cell-free syntheticbiochemistry route for raspberry ketone production atindustrial scale, Vaishali Thaore
Moore SJ, MacDonald JT, Wienecke S, et al., 2018, Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 115, Pages: E4340-E4349, ISSN: 0027-8424
Salih O, He S, Planamente S, et al., 2018, Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa, STRUCTURE, Vol: 26, Pages: 329-+, ISSN: 0969-2126
Hazel P, Kroll SHB, Bondke A, et al., 2018, Inhibitor Selectivity for Cyclin-Dependent Kinase7: A Structural, Thermodynamic, and Modelling Study (vol 12, pg 372, 2017), CHEMMEDCHEM, Vol: 13, Pages: 207-207, ISSN: 1860-7179
Lai H-E, Moore S, Polizzi K, et al., 2018, EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology., Pages: 429-444
Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.
Lai H-E, Obled AMC, Chee SM, et al., 2017, A GenoChemetic strategy for derivatization of the violacein natural product scaffold
Integrating synthetic chemistry with synthetic biology allows rapid access to xenobiotic compounds which may provide improved therapeutic activity. By supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with eight tryptophan substrate analogues or tryptophan halogenase RebH in vivo , 68 new-to-nature analogues of violacein were generated. Furthermore, 20 new derivatives were generated from brominated analogues via Suzuki-Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein, we demonstrate a flexible and rapid approach to access diverse chemical space that can be applied to a wide range of natural product scaffolds.
Wen KY, Cameron L, Chappell J, et al., 2017, A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples., ACS Synthetic Biology, Vol: 6, Pages: 2293-2301, ISSN: 2161-5063
Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.
Stach L, Freemont PS, 2017, The AAA+ ATPase p97, a cellular multitool, BIOCHEMICAL JOURNAL, Vol: 474, Pages: 2953-2976, ISSN: 0264-6021
Smith WD, Bardin E, Cameron L, et al., 2017, Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis, FEMS MICROBIOLOGY LETTERS, Vol: 364, ISSN: 0378-1097
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: 0300-5127
Goers L, Ainsworth C, Goey CH, et al., 2017, Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production, BIOTECHNOLOGY AND BIOENGINEERING, Vol: 114, Pages: 1290-1300, ISSN: 0006-3592
McClymont DW, Freemont PS, 2017, With all due respect to Maholo, lab automation isn't anthropomorphic, NATURE BIOTECHNOLOGY, Vol: 35, Pages: 312-314, ISSN: 1087-0156
Moore SJ, Lai H-E, Needham H, et al., 2017, Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool, BIOTECHNOLOGY JOURNAL, Vol: 12, ISSN: 1860-6768
Hazel P, Kroll SHB, Bondke A, et al., 2017, Inhibitor Selectivity for Cyclin-Dependent Kinase7: AStructural, Thermodynamic, and Modelling Study, CHEMMEDCHEM, Vol: 12, Pages: 372-380, ISSN: 1860-7179
Webb AJ, Kelwick R, Freemont PS, 2017, Opportunities for applying whole-cell bioreporters towards parasite detection, MICROBIAL BIOTECHNOLOGY, Vol: 10, Pages: 244-249, ISSN: 1751-7915
, 2017, Synthesising, Biologist, Vol: 64, Pages: 22-25, ISSN: 0006-3347
Kelwick R, 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: 1096-7176
MacDonald JT, Freemont PS, 2016, Computational protein design with backbone plasticity, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 44, Pages: 1523-1529, ISSN: 0300-5127
Moore SJ, Lai H-E, Kelwick RJR, et al., 2016, EcoFlex: A Multifunctional MoClo Kit for E-coli Synthetic Biology, ACS SYNTHETIC BIOLOGY, Vol: 5, Pages: 1059-1069, ISSN: 2161-5063
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: 0027-8424
Schuster CF, Bellows LE, Tosi T, et al., 2016, The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus, SCIENCE SIGNALING, Vol: 9, ISSN: 1945-0877
Planamente S, Salih O, Manoli E, et al., 2016, TssA forms a gp6-like ring attached to the type VI secretion sheath, EMBO JOURNAL, Vol: 35, Pages: 1613-1627, ISSN: 0261-4189
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