Publications
325 results found
Mercer T, Almond N, Chain P, et al., 2021, A roadmap to better COVID-19 testing from the Coronavirus Standards Working Group
<jats:title>Abstract</jats:title> <jats:p>Testing has been central to our response to the COVID-19 pandemic. However, the accuracy of testing relies on standards, including reference materials, proficiency testing schemes, and information and reporting guidelines. The use of standards is a simple, inexpensive, and effective method to ensure reliable test results that inform clinical and public health decisions. Here we describe the central role of standards during the COVID-19 pandemic, where they have enabled population-scale screening, genomic surveillance and measures of immune protection measures. Given these benefits, the Coronavirus Standards Working Group (CSWG) was formed to coordinate standards in SARS-CoV-2 testing. This network of scientists has developed best-practices, reference materials, and conducted proficiency studies to harmonize laboratory performance. We propose that this coordinated development of standards should be prioritized as a key early step in the public health response to future pandemics that is necessary for reliable, large-scale testing for infectious disease.</jats:p>
Farzaneh T, Freemont PS, 2021, Biofoundries are a nucleating hub for industrial translation, SYNTHETIC BIOLOGY, Vol: 6, Pages: 1-6
Pathania M, Tosi T, Millership C, et al., 2021, Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM, Journal of Biological Chemistry, Vol: 297, Pages: 1-15, ISSN: 0021-9258
Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.
Crone M, Randell P, Herm Z, et al., 2021, Rapid design and implementation of an adaptive pooling workflow for SARS-CoV-2 testing in an NHS diagnostic laboratory: a proof-of-concept study, Wellcome Open Research, Vol: 6, Pages: 1-13, ISSN: 2398-502X
Background: Diagnostic laboratories are currently required to provide routine testing of asymptomatic staff and patients as a part of their clinical screening for SARS-CoV-2 infection. However, these cohorts display very different disease prevalence from symptomatic individuals and testing capacity for asymptomatic screening is often limited. Group testing is frequently proposed as a possible solution to address this; however, proposals neglect the technical and operational feasibility of implementation in a front-line diagnostic laboratory.Methods: Between October and December 2020, as a seven-week proof of concept, we took into account scientific, technical and operational feasibility to design and implement an adaptive pooling strategy in an NHS diagnostic laboratory in London (UK). We assessed the impact of pooling on analytical sensitivity and modelled the impact of prevalence on pooling strategy. We then considered the operational constraints to model the potential gains in capacity and the requirements for additional staff and infrastructure. Finally, we developed a LIMS-agnostic laboratory automation workflow and software solution and tested the technical feasibility of our adaptive pooling workflow.Results: First, we determined the analytical sensitivity of the implemented SARS-CoV-2 assay to be 250 copies/mL. We then determined that, in a setting with limited analyser capacity, the testing capacity could be increased by two-fold with pooling, however, in a setting with limited reagents, this could rise to a five-fold increase. These capacity increases could be realized with modest additional resource and staffing requirements whilst utilizing up to 76% fewer plastic consumables and 90% fewer reagents. Finally, we successfully implemented a plate-based pooling workflow and tested 920 patient samples using the reagents that would usually be required to process just 222 samples.Conclusions: Adaptive pooled testing is a scientifically, technically and operatio
Toh M, Chengan K, Hanson T, et al., 2021, A High-Yield <i>Streptomyces</i> Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
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- Citations: 3
Moore S, Tosi T, Bell D, et al., 2021, High-yield ‘one-pot’ biosynthesis of raspberry ketone, a high-value fine chemical, Synthetic Biology, Vol: 6, Pages: 1-8, ISSN: 2397-7000
Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction with a yield of ~1-4 mg kg-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L-1. Herein, we report an efficient cell-free strategy to probe a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one (HBA), into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalysed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a ‘one-pot’ cell-free reaction to produce raspberry ketone at high-yield (61 mg L-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.
Moore SJ, Hleba YB, Bischoff S, et al., 2021, Refactoring of a synthetic raspberry ketone pathway with EcoFlex (vol 20, 116, 2021), Microbial Cell Factories, Vol: 20, Pages: 1-2, ISSN: 1475-2859
Rowan AG, May P, Badhan A, et al., 2021, Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control., Journal of Virological Methods, Vol: 294, Pages: 1-7, ISSN: 0166-0934
There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4% of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.
Garenne D, Haines MC, Romantseva EF, et al., 2021, Cell-free gene expression, NATURE REVIEWS METHODS PRIMERS, Vol: 1
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- Citations: 39
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.
Freemont P, 2021, Refactoring of a synthetic raspberry ketone pathway with EcoFlex, Microbial Cell Factories, Vol: 20, Pages: 1-11, ISSN: 1475-2859
Background A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit.Results By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture.Conclusions Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.
Crone M, Randell P, Herm Z, et al., 2021, Design and Implementation of An Adaptive Pooling Workflow for SARS-CoV-2 Testing in an NHS Diagnostic Laboratory
Moore SJ, Lai H-E, Chee S-M, et al., 2021, A <i>Streptomyces venezuelae</i> Cell-Free Toolkit for Synthetic Biology, ACS SYNTHETIC BIOLOGY, Vol: 10, Pages: 402-411, ISSN: 2161-5063
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- Citations: 22
Wu C-H, Rismondo J, Morgan RML, et al., 2021, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Journal of Biological Chemistry, Vol: 296, Pages: 1-14, ISSN: 0021-9258
UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.
Lossi NS, Manoli E, Foerster A, et al., 2021, The HsiB1C1 (TssB-TssC) complex of the pseudomonas aeruginosa Type VI secretion system forms a bacteriophage tail sheathlike structure, Journal of Biological Chemistry, Vol: 288, Pages: 7536-7548, ISSN: 0021-9258
Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ∼100 Å, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 Å. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs.
Young R, Haines M, Storch M, et al., 2021, Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly, Metabolic Engineering, Vol: 63, Pages: 81-101, ISSN: 1096-7176
Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.
Wu C-H, Rismondo J, Morgan RML, et al., 2020, <i>Bacillus subtilis</i>YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth
<jats:title>Abstract</jats:title><jats:p>UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In<jats:italic>Bacillus subtilis</jats:italic>168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The<jats:italic>B. subtilis</jats:italic>UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and<jats:italic>gtaB</jats:italic>mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in<jats:italic>B. subtilis</jats:italic>. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated<jats:italic>in vitro</jats:italic>using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a<jats:italic>B. subtilis gtaB</jats:italic>mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase<jats:italic>in vivo</jats:italic>. When wild-type and mutant<jats:italic>B. subtilis</jats:italic>strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing<jats:italic>yngB</jats:italic>and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues
Li H, Barnaghi P, Skillman S, et al., 2020, Machine learning for risk analysis of Urinary Tract Infection in people with dementia, Publisher: arXiv
The Urinary Tract Infections (UTIs) are one of the top reasons for unplannedhospital admissions in people with dementia, and if detected early, they can betimely treated. However, the standard UTI diagnosis tests, e.g. urine tests,will be only taken if the patients are clinically suspected of having UTIs.This causes a delay in diagnosis and treatment of the conditions and in somecases like people with dementia, the symptoms can be difficult to observe.Delay in detection and treatment of dementia is one of the key reasons forunplanned hospital admissions in people with dementia. To address these issues,we have developed a technology-assisted monitoring system, which is a Class 1medical device. The system uses off-the-shelf and low-cost in-home sensorydevices to monitor environmental and physiological data of people with dementiawithin their own homes. We have designed a machine learning model to use thedata and provide risk analysis for UTIs. We use a semi-supervised learningmodel which leverage the environmental data, i.e. the data collected from themotion sensors, smart plugs and network-connected body temperature monitoringdevices in the home, to detect patterns that can show the risk of UTIs. Sincethe data is noisy and partially labelled, we combine the neural networks andprobabilistic neural networks to train an auto-encoder, which is to extract thegeneral representation of the data. We will demonstrate our smart homemanagement by videos/online, and show how our model can pick up the UTI relatedpatterns.
Moore SJ, Lai H-E, Chee S-M, et al., 2020, A<i>Streptomyces venezuelae</i>Cell-Free Toolkit for Synthetic Biology
<jats:title>Abstract</jats:title><jats:p>Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The<jats:italic>Streptomyces</jats:italic>genus is a major source of natural products. To study enzymes and pathways from<jats:italic>Streptomyces</jats:italic>, we originally developed a homologous<jats:italic>Streptomyces</jats:italic>cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust<jats:italic>Streptomyces</jats:italic>TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of<jats:italic>Streptomyces</jats:italic>metabolic pathways in a single ‘one-pot’ reaction.</jats:p>
Crone MA, Priestman M, Ciechonska M, et al., 2020, A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics (vol 11, 4464, 2020), NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
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Crone M, Priestman M, Ciechonska M, et al., 2020, A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics, Nature Communications, Vol: 11, Pages: 1-11, ISSN: 2041-1723
The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.
Graham N, Junghans C, Downes R, et al., 2020, SARS-CoV-2 infection, clinical features and outcome of COVID-19 in United Kingdom nursing homes, Journal of Infection, Vol: 81, Pages: 411-419, ISSN: 0163-4453
OBJECTIVES: To understand SARS-Co-V-2 infection and transmission in UK nursing homes in order to develop preventive strategies for protecting the frail elderly residents. METHODS: An outbreak investigation involving 394 residents and 70 staff, was carried out in 4 nursing homes affected by COVID-19 outbreaks in central London. Two point-prevalence surveys were performed one week apart where residents underwent SARS-CoV-2 testing and had relevant symptoms documented. Asymptomatic staff from three of the four homes were also offered SARS-CoV-2 testing. RESULTS: Overall, 26% (95% CI 22 to 31) of residents died over the two-month period. All-cause mortality increased by 203% (95% CI 70 to 336) compared with previous years. Systematic testing identified 40% (95% CI 35 to 46) of residents as positive for SARS-CoV-2, and of these 43% (95% CI 34 to 52) were asymptomatic and 18% (95% CI 11 to 24) had only atypical symptoms; 4% (95% CI -1 to 9) of asymptomatic staff also tested positive. CONCLUSIONS: The SARS-CoV-2 outbreak in four UK nursing homes was associated with very high infection and mortality rates. Many residents developed either atypical or no discernible symptoms. A number of asymptomatic staff members also tested positive, suggesting a role for regular screening of both residents and staff in mitigating future outbreaks.
Graham NSN, Junghans C, Downes R, et al., 2020, SARS-CoV-2 infection, clinical features and outcome of COVID-19 in United Kingdom nursing homes
<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Objectives</jats:title><jats:p>To understand SARS-Co-V-2 infection and transmission in UK nursing homes in order to develop preventive strategies for protecting the frail elderly residents.</jats:p></jats:sec><jats:sec><jats:title>Design</jats:title><jats:p>An outbreak investigation.</jats:p></jats:sec><jats:sec><jats:title>Setting</jats:title><jats:p>4 nursing homes affected by COVID-19 outbreaks in central London.</jats:p></jats:sec><jats:sec><jats:title>Participants</jats:title><jats:p>394 residents and 70 staff in nursing homes.</jats:p></jats:sec><jats:sec><jats:title>Interventions</jats:title><jats:p>Two point-prevalence surveys one week apart where residents underwent SARS-CoV-2 testing and had relevant symptoms documented. Asymptomatic staff from three of the four homes were also offered SARS-CoV-2 testing.</jats:p></jats:sec><jats:sec><jats:title>Main outcome measures</jats:title><jats:p>All-cause mortality, and mortality attributed to COVID-19 on death certificates. Prevalence of SARS-CoV-2 infection and symptoms in residents and staff.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Overall, 26% (95% confidence interval 22 to 31) of residents died over the two-month period. All-cause mortality increased by 203% (95% CI 70 to 336). Systematic testing identified 40% (95% CI 35 to 46) of residents, of whom 43% (95% CI 34 to 52) were asymptomatic and 18% (95% CI 11 to 24) had atypical symptoms, as well as 4% (95% CI -1 to 9) of asymptomatic staff who tested positive for SARS-CoV-2.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The SARS-CoV-2 outbreak was associated with a ver
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.
Beal J, Goñi-Moreno A, Myers C, et al., 2020, The long journey towards standards for engineering biosystems: Are the Molecular Biology and the Biotech communities ready to standardise?, EMBO Reports, Vol: 21, Pages: 1-5, ISSN: 1469-221X
Synthetic biology needs to adopt sound scientific and industry-like standards in order to achieve its ambitious goals of efficient and accurate engineering of biological systems.
Crone MA, Priestman M, Ciechonska M, et al., 2020, A new role for Biofoundries in rapid prototyping, development, and validation of automated clinical diagnostic tests for SARS-CoV-2
<jats:title>Abstract</jats:title><jats:p>The SARS-CoV-2 pandemic has shown how the rapid rise in demand for patient and community sample testing, required for tracing and containing a highly infectious disease, has quickly overwhelmed testing capability globally. With most diagnostic infrastructure dependent on specialised instruments, their exclusive reagent supplies quickly become bottlenecks in times of peak demand, creating an urgent need for novel approaches to boost testing capacity. We address this challenge by refocusing the full synthetic biology stack available at the London Biofoundry onto the development of alternative patient sample testing pipelines. We present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled, and that accepts a diverse range of reagents. Using an in-house-generated, open-source, MS2-virus-like-particle-SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two novel detection assays based on CRISPR-Cas and Loop-mediated isothermal Amplification (LAMP) approaches. In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and benchmark SARS-CoV-2 detection in patient samples via RT-qPCR, CRISPR-Cas, and LAMP against clinical test sets. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs and now contributes to increased patient sample processing in the UK while we continue to refine and develop novel high-throughput diagnostic methods. Finally, our workflows and protocols can be quickly implemented and adapted by members of the Global Biofoundry Alliance and the wider scientific and medical diagnostics community.</jats:p>
Wilkinson MD, Lai H-E, Freemont PS, et al., 2020, A biosynthetic platform for antimalarial drug discovery, Antimicrobial Agents and Chemotherapy, Vol: 64, Pages: 1-9, ISSN: 0066-4804
Advances in synthetic biology have enabled production of a variety of compounds using bacteria as a vehicle for complex compound biosynthesis. Violacein, a naturally occurring indole pigment with antibiotic properties, can be biosynthetically engineered in Escherichia coli expressing its non-native synthesis pathway. To explore whether this synthetic biosynthesis platform could be used for drug discovery, here we have screened bacterially-derived violacein against the main causative agent of human malaria, Plasmodium falciparum. We show the antiparasitic activity of bacterially-derived violacein against the P. falciparum 3D7 laboratory reference strain as well as drug-sensitive and resistant patient isolates, confirming the potential utility of this drug as an antimalarial. We then screen a biosynthetic series of violacein derivatives against P. falciparum growth. The demonstrated varied activity of each derivative against asexual parasite growth points to potential for further development of violacein as an antimalarial. Towards defining its mode of action, we show that biosynthetic violacein affects the parasite actin cytoskeleton, resulting in an accumulation of actin signal that is independent of actin polymerization. This activity points to a target that modulates actin behaviour in the cell either in terms of its regulation or its folding. More broadly, our data show that bacterial synthetic biosynthesis could become a suitable platform for antimalarial drug discovery with potential applications in future high-throughput drug screening with otherwise chemically-intractable natural products.
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.
Stach L, Morgan RM, Makhlouf L, et al., 2020, Crystal structure of the catalytic D2 domain of the AAA+ ATPase p97 reveals a putative helical split-washer-type mechanism for substrate unfolding, FEBS Letters, Vol: 594, Pages: 933-943, ISSN: 0014-5793
Several pathologies have been associated with the AAA+ ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High‐resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high‐resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P61 symmetry suggests a mechanism for substrate unfolding by p97.
Kopniczky MB, Canavan C, McClymont DW, et al., 2020, Cell-free protein synthesis as a prototyping platform for mammalian synthetic biology, ACS Synthetic Biology, Vol: 9, Pages: 144-156, ISSN: 2161-5063
The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.
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