Publications
325 results found
Freemont PS, Friedman JM, Beese LS, et al., 2020, Cocrystal structure of an editing complex of klenow fragment with dna (3'-5' exonuclease dna polymerase protein-dna interaction x-ray crystallography/metal ion catalysis), Structural Insights into Gene Expression and Protein Synthesis, Pages: 240-244, ISBN: 9789811215858
High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3′ – 5′ exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3′ terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 Å away for the newly formed 3′ terminus. Since a 3′ terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.
de Martín Garrido N, Crone MA, Ramlaul K, et al., 2020, Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids, Molecular Microbiology, Vol: 113, Pages: 143-152, ISSN: 0950-382X
Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.
Webb AJ, Kelwick R, Wang Y, et al., 2019, AL-PHA beads: bioplastic-bsaed protease biosensors for global health, British Society for Parasitology Autumn Symposium, Belfast, UK
Lai H-E, Canavan C, Cameron L, et al., 2019, Synthetic biology and the United Nations, Trends in Biotechnology, Vol: 37, Pages: 1146-1151, ISSN: 0167-7799
Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).
Freemont P, 2019, Synthetic biology industry - Data-driven design is creating new opportunities in biotechnology., Emerging Topics in Life Sciences, Vol: 3, Pages: 651-657, ISSN: 2397-8554
Synthetic biology is a rapidly emerging interdisciplinary research field that is primarily built upon foundational advances in molecular biology combined with engineering design. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies. This has spurned a rapid growth in start-up companies and the new synthetic biology industry is growing rapidly, with start-up companies receiving ~$6.1B investment since 2015 and a global synthetic biology market value estimated to be $14B by 2026. Many of the new start-upscan be grouped within a multi-layer ‘technology stack’. The ‘stack’ comprises a number of technology layers which together can be applied to a diversity of new biotechnology applications like consumer biotechnology products and living therapies. The ‘stack’ also enables new commercial opportunities and value chains similar to the software design and manufacturing revolution of the 20th century. However, synthetic biology industry is at a crucial point, as it now requires recognisable commercial successes in order for the industry to expand and scale, in terms of investment and companies. However, such expansion may directly challenge the ethos of synthetic biology, in terms of open technology sharing and democratisation, which could by accident lead to multi-national corporations and technology monopolies similar to the existing biotechnology/biopharma industry.
Wood TE, Howard SA, Forster A, et al., 2019, The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology, Cell Reports, Vol: 29, Pages: 187-201.e7, ISSN: 2211-1247
The type VI secretion system (T6SS) is crucialin interbacterial competition and is avirulence determinant ofmany Gram-negative bacteria. Several T6SS effectorsarecovalently fused to secreted T6SS structural components such asthe VgrG spike for delivery into target cells.In Pseudomonas aeruginosa, theVgrG2b effector waspreviously proposedto mediatebacterial internalisation into eukaryotic cells. In this work, wefind that the VgrG2b C-terminal domain(VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein.We resolve thestructure of VgrG2bC-ter and confirm it is a member ofthezinc-metallopeptidasefamily of enzymes. We show that this effector causesmembrane blebbing atmidcell, whichsuggests a distincttype of T6SS-mediated growthinhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Ourstudyintroduces a further effector family to the T6SS arsenaland demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.
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.
Hillson N, Caddick M, Cai Y, et al., 2019, Building a global alliance of biofoundries (vol 10, 2040, 2019), Nature Communications, Vol: 10, Pages: 1-2, ISSN: 2041-1723
Rajakumar PD, Gower G, Suckling L, et al., 2019, Rapid prototyping platform for Saccharomyces cerevisiae using computer-aided genetic design enabled by parallel software and workcell platform development, Slas Technology, Vol: 24, Pages: 291-297, ISSN: 2472-6303
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.
Hillson N, Caddick M, Cai Y, et al., 2019, Building a global alliance of biofoundries, NATURE COMMUNICATIONS, Vol: 10, Pages: 1-4, ISSN: 2041-1723
Kelwick R, Webb AJ, Wang Y, et al., 2019, ISEV2019 Abstract Book. PT09.10: Protease biomarker detection using functionalised bioplastic-based biosensors, ISEV 2019, Publisher: Co-Action Publishing, ISSN: 2001-3078
Thaore V, Moore S, Polizzi K, et al., 2019, 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 tuneable limit of detection enables low-cost agglutination assays for medical diagnostic applications, ACS Sensors, Vol: 4, Pages: 370-378, ISSN: 2379-3694
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 can 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 straight-forward 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 nanbodies 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.
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, Pages: 1-28, ISSN: 1553-7366
c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
Exley K, Reynolds C, 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
BackgroundThe automation of modular cloning methodologies permits the assembly of many genetic designs. Utilising characterised biological parts aids in the design and redesign of genetic pathways. The characterisation information held on datasheets can be used to determine whether a biological part meets the design requirements. To manage the design of genetic pathways, researchers have turned to modelling-based computer aided design software tools.ResultAn automated workflow has been developed for the design and build of heterologous metabolic pathways. In addition, to demonstrate the powers of electronic datasheets we have developed software which can transfer part information from a datasheet to the Design of Experiment software JMP. To this end we were able to use Design of Experiment software to rationally design and test randomised samples from the design space of a lycopene pathway in E. coli. This pathway was optimised by individually modulating the promoter strength, RBS strength, and gene order targets.ConclusionThe use of standardised and characterised biological parts will empower a design-oriented synthetic biology for the forward engineering of heterologous expression systems. A Design of Experiment approach streamlines the design-build-test cycle to achieve optimised solutions in biodesign. Developed automated workflows provide effective transfer of information between characterised information (in the form of datasheets) and DoE software.
Appuswamy R, Brigand KL, Barbry P, et al., 2019, OligoArchive: Using DNA in the DBMS storage hierarchy, CIDR 2019
Appuswamy R, Lebrigand K, Barbry P, et al., 2019, Oligoarchive: Using DNA in the DBMS storage hierarchy
The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.
Webb AJ, Landeryou T, Kelwick R, et al., 2019, SPECIFIC NUCLEIC ACIDS LIGATION FOR DETECTION OF SCHISTOSOMES: SNAILS, 68th Annual Meeting of the American-Society-for-Tropical-Medicine-and-Hygiene (ASTMH), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 182-182, ISSN: 0002-9637
Appuswamy R, Lebrigand K, Barbry P, et al., 2019, Oligoarchive: Using DNA in the DBMS storage hierarchy
© 2019 Conference on Innovative Data Systems Research (CIDR). All rights reserved. The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.
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
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.
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, Publisher: American Chemical Society
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.
Thaore V, Moore S, Polizzi K, et al., 2018, Techno-economic evaluation of a cell-free syntheticbiochemistry route for raspberry ketone production atindustrial scale, Vaishali Thaore
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.
Hazel P, Kroll SHB, Bondke A, et al., 2018, Corrigendum: Inhibitor selectivity for cyclin-dependent kinase 7: a structural, thermodynamic, and modelling study, ChemMedChem, Vol: 13, Pages: 207-207, ISSN: 1860-7187
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.
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
<jats:title>Abstract</jats:title><jats:p>Natural products and their analogues are often challenging to synthesise due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an <jats:italic>Escherichia coli</jats:italic> strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan <jats:italic>in vitro</jats:italic> or tryptophan 7-halogenase RebH <jats:italic>in vivo</jats:italic>, 6 halogenated analogues of violacein or deoxyviolacein were generated, demonstrating promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein 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.</jats:p>
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.
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