25 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.
Braun L, Hazell L, Webb AJ, et al., 2020, Determining the viability of Schistosoma mansoni cercariae using fluorescence assays: an application for water treatment, PLOS Neglected Tropical Diseases, Vol: 14, ISSN: 1935-2727
Background:Schistosome cercariae are the human-infectious stage of the Schistosoma parasite. They are shed by snail intermediate hosts living in freshwater, and penetrate the skin of the human host to develop into schistosomes, resulting in schistosomiasis infection. Water treatment (e.g. filtration or chlorination) is one way of cutting disease transmission; it kills or removes cercariae to provide safe water for people to use for activities such as bathing or laundry as an alternative to infested lakes or rivers. At present, there is no standard method for assessing the effectiveness of water treatment processes on cercariae. Examining cercarial movement under a microscope is the most common method, yet it is subjective and time-consuming. Hence, there is a need to develop and verify accurate, high-throughput assays for quantifying cercarial viability.Method:We tested two fluorescence assays for their ability to accurately determine cercarial viability in water samples, using S. mansoni cercariae released from infected snails in the Schistosomiasis Collection at the Natural History Museum, London. These assays consist of dual stains, namely a vital and non-vital dye; fluorescein diacetate (FDA) and Hoechst, and FDA and Propidium Iodide. We also compared the results of the fluorescence assays to the viability determined by microscopy.Conclusion:Both fluorescence assays can detect the viability of cercariae to an accuracy of at least 92.2% ± 6.3%. Comparing the assays to microscopy, no statistically significant difference was found between the method’s viability results. However, the fluorescence assays are less subjective and less time-consuming than microscopy, and therefore present a promising method for quantifying the viability of schistosome cercariae in water samples.
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
Kelwick R, Ricci L, Chee SM, et al., 2017, Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics
<jats:title>ABSTRACT</jats:title><jats:p>The polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the polyhydroxyalkanoates biosynthetic pathways. Here we have developed several <jats:italic>Escherichia coli</jats:italic> cell-free transcription-translation-based systems for <jats:italic>in vitro</jats:italic> prototyping of polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising <jats:italic>in vivo</jats:italic> polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3μM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a <jats:italic>Clostridium propionicum</jats:italic> propionyl CoA transferase enzyme that can recycle Acetyl-
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
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.
Percy M, Karinou E, Webb A, et al., 2016, Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications, Journal of Bacteriology, Vol: 198, Pages: 2029-2042, ISSN: 1098-5530
Listeria monocytogenes is a food-borne Gram-positive bacterial pathogen and many of its virulence factors are either secreted proteins, or proteins covalently or non-covalently-attached to the cell wall. Previous work has indicated that non-covalently-attached proteins with GW domains are retained in the cell wall by binding to the cell wall polymer lipoteichoic acid (LTA). LTA is a glycerolphosphate polymer, which is modified in L. monocytogenes with galactose and D-alanine residues. We identified Lmo0933 as the cytoplasmic glycosyltransferase required for the LTA glycosylation process and renamed the protein GtlA for glycosyltransferase LTA A. Using L. monocytogenes mutants lacking galactose or D-alanine modifications or the complete LTA polymer, we show that GW-domain proteins are still retained within the cell wall, indicating that other cell wall polymers are involved in the retention of GW-domain proteins. Further experiments reveal peptidoglycan as the binding receptor as a purified GW domainfusion protein can bind to L. monocytogenes cells lacking wall teichoic acid (WTA) as well as purified peptidoglycan derived from a wild-type or WTA-negative strain. With this, we not only identified the first enzyme involved in the LTA glycosylation process, but we also provide new insight into the binding mechanism of non-covalently attached cell wall proteins.
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, 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, MacDonald JT, Webb AJ, et al., 2014, Developments in the Tools and Methodologies of Synthetic Biology, Frontiers in Bioengineering and Biotechnology, Vol: 2
Webb AJ, Karatsa-Dodgson M, Grundling A, 2009, Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes, MOLECULAR MICROBIOLOGY, Vol: 74, Pages: 299-314, ISSN: 0950-382X
Basavanna S, Khandavilli S, Yuste J, et al., 2009, Screening of Streptococcus pneumoniae ABC Transporter Mutants Demonstrates that LivJHMGF, a Branched-Chain Amino Acid ABC Transporter, Is Necessary for Disease Pathogenesis, INFECTION AND IMMUNITY, Vol: 77, Pages: 3412-3423, ISSN: 0019-9567
Webb AJ, Homer KA, Hosie AHF, 2008, Two closely related ABC transporters in Streptococcus mutans are involved in disaccharide and/or oligosaccharide uptake, JOURNAL OF BACTERIOLOGY, Vol: 190, Pages: 168-178, ISSN: 0021-9193
Webb AJ, Homer KA, Hosie AHF, 2007, A phosphoenolpyruvate-dependent phosphotransferase system is the principal maltose transporter in Streptococcus mutans, JOURNAL OF BACTERIOLOGY, Vol: 189, Pages: 3322-3327, ISSN: 0021-9193
Webb AJ, Hosie AHF, 2006, A member of the second carbohydrate uptake subfamily of ATP-binding cassette transporters is responsible for ribonucleoside uptake in Streptococcus mutans, JOURNAL OF BACTERIOLOGY, Vol: 188, Pages: 8005-8012, ISSN: 0021-9193
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