28 results found
Kelwick R, Webb A, Heliot A, et al., 2023, Opportunities to accelerate extracellular vesicle research with cell-free synthetic biology, Journal of Extracellular Biology, Vol: 2, Pages: 1-12, ISSN: 2768-2811
Extracellular vesicles (EVs) are lipid-membrane nanoparticles that are shed or secreted by many different cell types. The extracellular vesicle (EV) research community has rapidly expanded in recent years and are leading efforts to deepen our understanding of EV biological functions in human physiology and pathology. These insights are also providing a foundation on which future EV-based diagnostics and therapeutics are poised to positively impact human health. However, current limitations in our understanding of EV heterogeneity, cargo loading mechanisms and the nascent development of EV metrology are all areas that have been identified as important scientific challenges. The field of synthetic biology is also contending with the challenge of understanding biological complexity as it seeks to combine multidisciplinary scientific knowledge with engineering principles, to build useful and robust biotechnologies in a responsible manner. Within this context, cell-free systems have emerged as a powerful suite of in vitro biotechnologies that can be employed to interrogate fundamental biological mechanisms, including the study of aspects of EV biogenesis, or to act as a platform technology for medical biosensors and therapeutic biomanufacturing. Cell-free gene expression (CFE) systems also enable in vitro protein production, including membrane proteins, and could conceivably be exploited to rationally engineer, or manufacture, EVs loaded with bespoke molecular cargoes for use in foundational or translational EV research. Our pilot data herein, also demonstrates the feasibility of cell-free EV engineering. In this perspective we discuss the opportunities and challenges for accelerating EV research and healthcare applications with cell-free synthetic biology.
Kelwick R, Webb A, Freemont P, 2023, Opportunities for engineering Outer Membrane Vesicles (OMVs) using synthetic biology approaches, Extracellular Vesicles and Circulating Nucleic Acids, Vol: 4, Pages: 255-261, ISSN: 2767-6641
Gram-negative bacteria naturally shed lipid vesicles, which contain complex molecular cargoes, from their outer membrane. These outer membrane vesicles (OMVs) have important biological functions relating to microbial stress responses, microbiome regulation, and host-pathogen interactions. OMVs are also attractive vehicles for delivering drugs, vaccines, and other therapeutic agents because of their ability to interact with host cells and their natural immunogenic properties. OMVs are also set to have a positive impact on other biotechnological and medical applications including diagnostics, bioremediation, and metabolic engineering. We envision that the field of synthetic biology offers a compelling opportunity to further expand and accelerate the foundational research and downstream applications of OMVs in a range of applications including the provision of OMV-based healthcare technologies. In our opinion, we discuss how current and potential future synergies between OMV research and synthetic biology approaches might help to further accelerate OMV research and real-world applications for the benefit of animal and human health.
Webb A, Allan F, Kelwick R, et al., 2022, Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS, PLOS Neglected Tropical Diseases, Vol: 16, Pages: 1-19, ISSN: 1935-2727
Schistosomiasis, also known as bilharzia or snail fever, is a debilitating neglected tropical disease (NTD), caused by parasitic trematode flatworms of the genus Schistosoma, that has an annual mortality rate of 280,000 people in sub-Saharan Africa alone. Schistosomiasis is transmitted via contact with water bodies that are home to the intermediate host snail which shed the infective cercariae into the water. Schistosome lifecycles are complex, and while not all schistosome species cause human disease, endemic regions also typically feature animal infecting schistosomes that can have broader economic and/or food security implications. Therefore, the development of species-specific Schistosoma detection technologies may help to inform evidence-based local environmental, food security and health systems policy making. Crucially, schistosomiasis disproportionally affects low- and middle-income (LMIC) countries and for that reason, environmental screening of water bodies for schistosomes may aid with the targeting of water, sanitation, and hygiene (WASH) interventions and preventive chemotherapy to regions at highest risk of schistosomiasis transmission, and to monitor the effectiveness of such interventions at reducing the risk over time. To this end, we developed a DNA-based biosensor termed Specific Nucleic AcId Ligation for the detection of Schistosomes or ‘SNAILS’. Here we show that ‘SNAILS’ enables species-specific detection from genomic DNA (gDNA) samples that were collected from the field in endemic areas.
Kelwick RJR, Webb AJ, Wang Y, et al., 2021, AL-PHA beads: bioplastic-based protease biosensors for global health applications, Materials Today, Vol: 47, Pages: 25-37, ISSN: 1369-7021
Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads – resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.
Kelwick RJR, Webb AJ, Wang Y, et al., 2020, AL-PHA beads: bioplastic-based protease biosensors for global health applications
<jats:title>ABSTRACT</jats:title><jats:p>Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads - resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from <jats:italic>Schistosoma mansoni</jats:italic>-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.</jats:p>
Kelwick R, Webb A, Freemont P, 2020, Biological materials: the next frontier for cell-free synthetic biology, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
Advancements in cell-free synthetic biology are enabling innovations in sustainable biomanufacturing, that may ultimately shift the global manufacturing paradigm toward localized and ecologically harmonized production processes. Cell-free synthetic biology strategies have been developed for the bioproduction of fine chemicals, biofuels and biological materials. Cell-free workflows typically utilize combinations of purified enzymes, cell extracts for biotransformation or cell-free protein synthesis reactions, to assemble and characterize biosynthetic pathways. Importantly, cell-free reactions can combine the advantages of chemical engineering with metabolic engineering, through the direct addition of co-factors, substrates and chemicals –including those that are cytotoxic. Cell-free synthetic biology is also amenable to automatable design cycles through which an array of biological materials and their underpinning biosynthetic pathways can be tested and optimized in parallel. Whilst challenges still remain, recent convergences between the materials sciences and these advancements in cell-free synthetic biology enable new frontiers for materials research.
Moore SJ, Lai H-E, Kelwick RJR, et al., 2020, Correction to EcoFlex: a multifunctional MoClo kit for E. coli synthetic biology., ACS Synthetic Biology, ISSN: 2161-5063
It has been brought to our attention that the original article contains a typographical error within Figure 1B, part ii. One of the 4-bp overhangs reads “GGAC” and should instead be “GTAC”, as is consistent throughout the original manuscript and deposited AddGene sequences.
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
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.
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
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
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
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.
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
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.
Kelwick RJR, 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: 1096-7184
Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.
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
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses anddeath. Rapid and cost-effective approaches to detect parasites are needed, especially inresource-limited settings. A common signature of parasitic diseases is the release of specificproteases by the parasites at multiple stages during their life cycles. To this end, weengineered several modular Escherichia coli and Bacillus subtilis whole-cell-basedbiosensors which incorporate an interchangeable protease recognition motif into theirdesigns. Herein, we describe how several of our engineered biosensors have been applied todetect the presence and activity of elastase, an enzyme released by the cercarial larvae stageof Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes areresponsible for the infection of an estimated 200 million people worldwide. Since ourbiosensors are maintained in lyophilised cells, they could be applied for the detection of S.mansoni and other parasites in settings without reliable cold chain access.
Kelwick R, Webb AJ, Macdonald JT, et al., 2016, Development of a bacillus subtilis cell-free transcriptiontranslation system
Kelwick R, Bowater L, Yeoman KH, et al., 2015, Promoting microbiology education through the iGEM synthetic biology competition, FEMS Microbiology Letters, Vol: 362, ISSN: 0378-1097
Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.
Kelwick R, Desanlis I, Wheeler GN, et al., 2015, The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family., Genome Biology, Vol: 16, Pages: 113-113, ISSN: 1474-760X
The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future.
Kelwick R, Kopniczky M, Bower I, et al., 2015, A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli, PLOS ONE, Vol: 10, ISSN: 1932-6203
Kelwick R, Wagstaff L, Decock J, et al., 2015, Metalloproteinase-Dependent and -Independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15, International Journal of Cancer, Vol: 136, Pages: E14-E26, ISSN: 0020-7136
The ADAMTS proteinases are a family of secreted, matrix-Associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.
Kelwick R, Wagstaff L, Decock J, et al., 2015, Metalloproteinase-dependent and -independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15, INTERNATIONAL JOURNAL OF CANCER, Vol: 136, Pages: E14-E26, ISSN: 0020-7136
Kelwick R, MacDonald JT, Webb AJ, et al., 2014, Developments in the Tools and Methodologies of Synthetic Biology, Frontiers in Bioengineering and Biotechnology, Vol: 2
Dobson R, Hicks JE, Gritton R, et al., 2014, Characterization of a rationally engineered nitric oxide, nitrate and nitrite biosensor linked to a hybrid bacterial-mammalian promoter, Figshare
Synthetic biology is principally concerned with the rational design and engineering of biological systems that serve useful applied purposes. Biosensors are of particular interest to the field since they serve a broad array of applications, such as medical devices, environmental sensors for the detection of contaminants, toxins or pathogens or in metabolic engineering, to monitor product formation. In this study, we describe the characterization of a family of four nitric oxide, nitrate and nitrite wholecell biosensors that are based upon a hybrid bacterial-mammalian promoter design. The hybrid design of the synthetic promoter has been engineered for the detection of these nitrogenous species across both bacterial (Escherichia coli) and mammalian systems (MCF-7). As such, these biosensors may be useful across applications as diverse as cancer therapeutics and the agricultural monitoring of nitrates and nitrites in fertiliser treated soil. Qualitative and quantitative analysis of these biosensors in E. coli confirmed that all four biosensor designs (termed BM_eCFP, BM_mRFP, MB_eCFP and MB_mRFP) were able to quantitatively detect 5-20 mM of potassium nitrate. In summary, these pilot data suggest that, with further characterisation, this family of biosensors will be able to assess nitrogenous species present within both bacterial (E. coli) and mammalian systems (MCF7).
Wagstaff L, Kelwick R, Decock J, et al., 2011, The roles of ADAMTS metalloproteinases in tumorigenesis and metastasis, FRONTIERS IN BIOSCIENCE-LANDMARK, Vol: 16, Pages: 1861-1872, ISSN: 1093-9946
Wagstaff L, Kelwick R, Decock J, et al., 2010, ADAMTS15 metalloproteinase inhibits breast cancer cell migration, Breast Cancer Research, Vol: 12
Kelwick R, The role of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-15 (ADAMTS-15) in Breast Cancer
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