246 results found
Thaore V, Moore S, Polizzi K, et al., Cell-free multi-enzyme system for the industrial production of fine chemicals, Chemical Engineering Day UK 2019
Suckling L, McFarlane C, Sawyer C, et al., 2019, Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation, Synthetic and Systems Biotechnology, Vol: 4, Pages: 57-66, ISSN: 2405-805X
High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.
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, Vol: 4, Pages: 370-378
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.
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 SENSORS, Vol: 4, Pages: 370-378, ISSN: 2379-3694
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
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
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.
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
Webb A, Allan F, Kelwick R, et al., 2018, PROTEASE-BASED BIOREPORTERS FOR THE DETECTION OF <it>SCHISTOSOMA CERCARIAE</it>, 67th Annual Meeting of the American-Society-of-Tropical-Medicine-and-Hygiene (ASTHM), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 191-191, ISSN: 0002-9637
Kelwick R, Ricci L, Chee SM, et al., 2018, Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics, Synthetic Biology, Vol: 3
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
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
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
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
Freemont P, 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
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