239 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.
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 Res
NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3'-phosphate and other 3'-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3'-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3'-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3'-phosphatase NExo bound to DNA in the presence and absence of a 3'-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3'-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.
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.
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, Chee SM, Morgan M, et al., 2017, A semi-synthetic strategy for derivatization of the violacein natural product scaffold
The next frontier in drug discovery could be the semi-synthesis of non-natural, xenobiotic compounds combining both natural product biosynthesis and synthetic chemistry. However, the required tools and underlying engineering principles are yet to be fully understood. One way to investigate non-natural product biosynthesis is to probe the substrate promiscuity of a clinically relevant biosynthesis pathway. Violacein is a bisindole compound produced by the VioABCDE biosynthesis pathway using L-tryptophan as the starting substrate. Previous studies have shown that violacein exhibits antimicrobial properties, and synthetic analogues of violacein might give rise to new targets for therapeutic development to combat antimicrobial resistance. By adding seven types of tryptophan analogues available commercially, 62 new violacein or deoxyviolacein analogues were generated with a synthetic violacein biosynthesis pathway expressed in Escherichia coli , demonstrating the promiscuity of violacein biosynthesis enzymes. Growth inhibition assays against Bacillus subtilis , a Gram-positive bacterium, were carried out to measure growth inhibitory activity of violacein analogues compared to violacein. In addition, we show that four new 7-chloro analogues of violacein or deoxyviolacein can be generated in vivo by combining the rebeccamycin and violacein biosynthesis pathways and purified 7-chloro violacein was found to have similar growth inhibitory activity compared to violacein. Structural studies of VioA revealed active site residues that are important for catalytic activity, and further pathway recombination with VioA homologues in related bisindole pathways may lead to more efficient enzymes that would accept tryptophan analogues more readily.
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
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
Filloux A, Freemont P, 2016, Baseplates in contractile machines, NATURE MICROBIOLOGY, Vol: 1
Chambers S, Kitney R, Freemont P, 2016, The Foundry: the DNA synthesis and construction Foundry at Imperial College, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 44, Pages: 687-688, ISSN: 0300-5127
Florea M, Hagemann H, Santosa G, et al., 2016, Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 113, Pages: E3431-E3440, ISSN: 0027-8424
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
Florea M, Reeve B, Abbott J, et al., 2016, Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582, SCIENTIFIC REPORTS, Vol: 6, ISSN: 2045-2322
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