315 results found
Hoose A, Vellacott R, Storch M, et al., 2023, DNA synthesis technologies to close the gene writing gap., Nature Reviews Chemistry, Pages: 1-18, ISSN: 2397-3358
Synthetic DNA is of increasing demand across many sectors of research and commercial activities. Engineering biology, therapy, data storage and nanotechnology are set for rapid developments if DNA can be provided at scale and low cost. Stimulated by successes in next generation sequencing and gene editing technologies, DNA synthesis is already a burgeoning industry. However, the synthesis of >200 bp sequences remains unaffordable. To overcome these limitations and start writing DNA as effectively as it is read, alternative technologies have been developed including molecular assembly and cloning methods, template-independent enzymatic synthesis, microarray and rolling circle amplification techniques. Here, we review the progress in developing and commercializing these technologies, which are exemplified by innovations from leading companies. We discuss pros and cons of each technology, the need for oversight and regulatory policies for DNA synthesis as a whole and give an overview of DNA synthesis business models.
Haines MC, Carling B, Marshall J, et al., 2022, basicsynbio and the BASIC SEVA collection: software and vectors for an established DNA assembly method, Synthetic Biology, Vol: 7, ISSN: 2397-7000
Crone MA, Freemont PS, 2022, Simple low-cost production of DNA MS2 virus-like particles as molecular diagnostic controls, GEN Biotechnology, Vol: 1, Pages: 496-503, ISSN: 2768-1556
Suitable controls are integral for the validation and continued quality assurance of diagnostic workflows. Plasmids, DNA, or in vitro transcribed RNA are often used to validate novel diagnostic workflows, however, they are poorly representative of clinical samples. RNA phage virus-like particles (VLPs) packaged with exogenous RNA have been used in clinical diagnostics as workflow controls, serving as surrogates for infectious viral particles. Comparable controls for DNA viruses are more challenging to produce, with analogous DNA phages being infectious and packaging of DNA within RNA phages requiring complex purification procedures and expensive chemical linkers. We present a simple and inexpensive method to produce Emesvirus zinderi (MS2) VLPs, packaged with DNA, that makes use of affinity chromatography for purification and enzymatic production of exogenous DNA suitable for packaging. The produced VLPs were packaged with hepatitis B virus DNA and were then quantified using droplet digital PCR and calibrated against the WHO international standard using a commercial assay in an accredited clinical laboratory.
Patchsung M, Homchan A, Aphicho K, et al., 2022, A multiplexed Cas13-based assay with point-of-care attributes for simultaneous COVID-19 diagnosis and variant surveillance, The CRISPR Journal, Pages: 1-17, ISSN: 2573-1599
Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection—such as multiplexed detection for viral variant surveillance—may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)—including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)—all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool—CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured—may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.
Moore SJJ, Lai H-E, Li J, et al., 2022, Streptomyces cell-free systems for natural product discovery and engineering, NATURAL PRODUCT REPORTS, ISSN: 0265-0568
Mercer T, Almond N, Crone MA, et al., 2022, The Coronavirus Standards Working Group's roadmap for improved population testing, NATURE BIOTECHNOLOGY, Vol: 40, Pages: 1563-1568, ISSN: 1087-0156
Hakki S, Zhou J, Jonnerby J, et al., 2022, Onset and window of SARS-CoV-2 infectiousness and temporal correlation with symptom onset: a prospective, longitudinal, community cohort study, The Lancet Respiratory Medicine, Vol: 10, Pages: 1061-1073, ISSN: 2213-2600
BACKGROUND: Knowledge of the window of SARS-CoV-2 infectiousness is crucial in developing policies to curb transmission. Mathematical modelling based on scarce empirical evidence and key assumptions has driven isolation and testing policy, but real-world data are needed. We aimed to characterise infectiousness across the full course of infection in a real-world community setting. METHODS: The Assessment of Transmission and Contagiousness of COVID-19 in Contacts (ATACCC) study was a UK prospective, longitudinal, community cohort of contacts of newly diagnosed, PCR-confirmed SARS-CoV-2 index cases. Household and non-household exposed contacts aged 5 years or older were eligible for recruitment if they could provide informed consent and agree to self-swabbing of the upper respiratory tract. The primary objective was to define the window of SARS-CoV-2 infectiousness and its temporal correlation with symptom onset. We quantified viral RNA load by RT-PCR and infectious viral shedding by enumerating cultivable virus daily across the course of infection. Participants completed a daily diary to track the emergence of symptoms. Outcomes were assessed with empirical data and a phenomenological Bayesian hierarchical model. FINDINGS: Between Sept 13, 2020, and March 31, 2021, we enrolled 393 contacts from 327 households (the SARS-CoV-2 pre-alpha and alpha variant waves); and between May 24, 2021, and Oct 28, 2021, we enrolled 345 contacts from 215 households (the delta variant wave). 173 of these 738 contacts were PCR positive for more than one timepoint, 57 of which were at the start of infection and comprised the final study population. The onset and end of infectious viral shedding were captured in 42 cases and the median duration of infectiousness was 5 (IQR 3-7) days. Although 24 (63%) of 38 cases had PCR-detectable virus before symptom onset, only seven (20%) of 35 shed infectious virus presymptomatically. Symptom onset was a median of 3 days before both peak viral RNA and
Cordery R, Reeves L, Zhou J, et al., 2022, Transmission of SARS-CoV-2 by children to contacts in schools and households: a prospective cohort and environmental sampling study in London, The Lancet Microbe, Vol: 3, Pages: e814-e823, ISSN: 2666-5247
Background: Assessing transmission of SARS-CoV-2 by children in schools is of critical importance to inform public health action. We assessed frequency of acquisition of SARS-CoV-2 by contacts of pupils with COVID-19 in schools and households, and quantified SARS-CoV-2 shed into air and onto fomites in both settings.Methods: Incidents involving exposure to at least one index pupil with COVID-19 in 8 schools were identified between October 2020-July 2021 (prevailing variants, original, alpha and delta). Weekly PCR testing for SARS-CoV-2 was undertaken on immediate classroom contacts (the “bubble”), non-bubble school contacts, and household contacts of index pupils, supported by genome sequencing, and on surface and air samples from school and home environments.Findings: Secondary transmission of SARS-CoV-2 was not detected in 28 bubble contacts, representing 10 bubble classes (participation rate 8.8%, IQR 4.6-15.3%). Across 8 non-bubble classes, 3/62 pupils tested positive but these were unrelated to the original index case (participation rate 22.5%, IQR 9.7-32.3%). All three were asymptomatic and tested positive in one setting on the same day. In contrast, secondary transmission to previously-negative household contacts from infected index pupils was 17.1% (6/35) rising to 27.7% (13/47) when considering all potentialinfections in household contacts. Environmental contamination with SARS-CoV-2 was rare in schools; fomite SARS-CoV-2 was identified in 4/189 (2.1%) samples in bubble classrooms, 2/127 (1.6%) samples in non-bubble classrooms, and 5/130 (3.8%) samples in washrooms. This contrasted with fomites in households, where SARS-CoV-2 was identified in 60/248 (24.2%) bedroom samples, 66/241 (27.4%) communal room samples, and 21/188 (11.2%) bathroom samples. Air sampling identified SARS-CoV-2 RNA in just 1/68 (1.5%) of school air samples, compared with 21/85 (24.7%) of air samples taken in homes.Interpretation: There was no evidence of large scale SARS-Co
Mosscrop L, Watber P, Elliot P, et al., 2022, Evaluation of the impact of pre-analytical conditions on sample stability for the detection of SARS-CoV-2 RNA, Journal of Virological Methods, Vol: 309, Pages: 1-5, ISSN: 0166-0934
Demand for accurate SARS-CoV-2 diagnostics is high. Most samples in the UK are collected in the community and rely on the postal service for delivery to the laboratories. The current recommendation remains that swabs should be collected in Viral Transport Media (VTM) and transported with a cold chain to the laboratory for RNA extraction and RT-qPCR. This is not always possible. We aimed to test the stability of SARS-CoV-2 RNA subjected to different pre-analytical conditions. Swabs were dipped into PBS containing cultured SARS-CoV-2 and placed in either a dry tube or a tube containing either normal saline or VTM. The tubes were then stored at different temperatures (20–50 °C) for variable periods (8 h to 5 days). Samples were tested by RT-qPCR targeting SARS-CoV-2 E gene. VTM outperformed swabs in saline and dry swabs in all conditions. Samples in VTM were stable, independent of a cold chain, for 5 days, with a maximum increase in cycle threshold (Ct) of 1.34 when held at 40 °C. Using normal saline as the transport media resulted in a loss of sensitivity (increased Ct) over time and with increasing temperature (up to 7.8 cycles compared to VTM). SARS-CoV-2 was not detected in 3/9 samples in normal saline when tested after 120 h incubation. Transportation of samples in VTM provides a high level of confidence in the results despite the potential for considerable, uncontrolled variation in temperature and longer transportation periods. False negative results may be seen after 96 h in saline and viral loads will appear lower.
Crone M, MacDonald J, Freemont P, et al., 2022, gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells, npj Systems Biology and Applications, Vol: 8, Pages: 1-7, ISSN: 2056-7189
Synthetic networks require complex intertwined genetic regulation often relying on transcriptional activation or repression of target genes. CRISPRi-based transcription factors facilitate the programmable modulation of endogenous or synthetic promoter activity and can be aided by using software to select appropriate gRNAs and limit non-specific gene modulation. Here, we develop a computational software pipeline, gDesigner, that enables the automated selection of orthogonal gRNAs with minimized off-target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. Our system expands the toolkit of mammalian synthetic promoters with a new complementary and orthogonal CRISPRi-based system, ultimately enabling the design of synthetic promoter libraries for multiplex gene perturbation that facilitate the understanding of complex cellular phenotypes.
Crone MA, Freemont PS, 2022, Simple, low-cost production of DNA MS2 virus-like particles as molecular diagnostic controls
<jats:title>Abstract</jats:title><jats:p>Suitable controls are integral for the validation and continued quality assurance of diagnostic workflows. Plasmids, DNA or <jats:italic>in vitro</jats:italic> transcribed RNA are often used to validate novel diagnostic workflows, however, they are poorly representative of clinical samples. RNA phage virus-like particles packaged with exogenous RNA have been used in clinical diagnostics as workflow controls, serving as surrogates for infectious viral particles. Comparable controls for DNA viruses are more challenging to produce, with analogous DNA phages being infectious and packaging of DNA within RNA phages requiring complex purification procedures and expensive chemical linkers. We present a simple and inexpensive method to produce MS2 virus-like particles, packaged with DNA, that makes use of affinity chromatography for purification and enzymatic production of exogenous DNA suitable for packaging. The produced virus-like particles were packaged with Hepatitis B Virus DNA and were then quantified using droplet digital PCR and calibrated against the WHO international standard using a commercial assay in an accredited clinical laboratory.</jats:p>
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(7):e0010632, 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.
Dixon TA, Freemont PS, Johnson RA, et al., 2022, A global forum on synthetic biology: the need for international engagement, NATURE COMMUNICATIONS, Vol: 13
- Author Web Link
- Citations: 3
Gil Rosa B, Akingbade OE, Guo X, et al., 2022, Multiplexed immunosensors for point-of-care diagnostic applications, Biosensors and Bioelectronics, Vol: 203, ISSN: 0956-5663
Accurate, reliable, and cost-effective immunosensors are clinically important for the early diagnosis and monitoring of progressive diseases, and multiplexed sensing is a promising strategy for the next generation of diagnostics. This strategy allows for the simultaneous detection and quantification of multiple biomarkers with significantly enhanced reproducibility and reliability, whilst requiring smaller sample volumes, fewer materials, and shorter average analysis time for individual biomarkers than individual tests. In this opinionated review, we compare different techniques for the development of multiplexed immunosensors. We review the state-of-the-art approaches in the field of multiplexed immunosensors using electrical, electrochemical, and optical methods. The barriers that prevent translating this sensing strategy into clinics are outlined together with the potential solutions. We also share our vision on how multiplexed immunosensors will continue their evolution in the coming years.
Wong G, Lim LR, Tan YQ, et al., 2022, Reconstituting the complete biosynthesis of D-lysergic acid in yeast, NATURE COMMUNICATIONS, Vol: 13
- Author Web Link
- Citations: 3
Singanayagam A, Hakki S, Dunning J, et al., 2022, Community transmission and viral load kinetics of the SARS-CoV-2 delta (B.1.617.2) variant in vaccinated and unvaccinated individuals in the UK: a prospective, longitudinal, cohort study., The Lancet. Infectious diseases, Vol: 22, Pages: 183-195, ISSN: 1473-3099
<h4>Background</h4>The SARS-CoV-2 delta (B.1.617.2) variant is highly transmissible and spreading globally, including in populations with high vaccination rates. We aimed to investigate transmission and viral load kinetics in vaccinated and unvaccinated individuals with mild delta variant infection in the community.<h4>Methods</h4>Between Sept 13, 2020, and Sept 15, 2021, 602 community contacts (identified via the UK contract-tracing system) of 471 UK COVID-19 index cases were recruited to the Assessment of Transmission and Contagiousness of COVID-19 in Contacts cohort study and contributed 8145 upper respiratory tract samples from daily sampling for up to 20 days. Household and non-household exposed contacts aged 5 years or older were eligible for recruitment if they could provide informed consent and agree to self-swabbing of the upper respiratory tract. We analysed transmission risk by vaccination status for 231 contacts exposed to 162 epidemiologically linked delta variant-infected index cases. We compared viral load trajectories from fully vaccinated individuals with delta infection (n=29) with unvaccinated individuals with delta (n=16), alpha (B.1.1.7; n=39), and pre-alpha (n=49) infections. Primary outcomes for the epidemiological analysis were to assess the secondary attack rate (SAR) in household contacts stratified by contact vaccination status and the index cases' vaccination status. Primary outcomes for the viral load kinetics analysis were to detect differences in the peak viral load, viral growth rate, and viral decline rate between participants according to SARS-CoV-2 variant and vaccination status.<h4>Findings</h4>The SAR in household contacts exposed to the delta variant was 25% (95% CI 18-33) for fully vaccinated individuals compared with 38% (24-53) in unvaccinated individuals. The median time between second vaccine dose and study recruitment in fully vaccinated contacts was longer for infected individuals (medi
Vickers CE, Freemont PS, 2022, Pandemic preparedness: synthetic biology and publicly funded biofoundries can rapidly accelerate response time COMMENT, NATURE COMMUNICATIONS, Vol: 13
- Author Web Link
- Citations: 3
Haines MC, Carling B, Marshall J, et al., 2022, basicsynbio and the BASIC SEVA collection: Software and vectors for an established DNA assembly method
<jats:title>Abstract</jats:title><jats:p>Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore design spaces and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use, while enabling simple hierarchical assemblies through an idempotent format. These features facilitate various applications including pathway engineering, ribosome binding site tuning, fusion protein engineering and multiplexed gRNA expression. In this work we present basicsynbio, an open-source software encompassing a Web App (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://basicsynbio.web.app/">https://basicsynbio.web.app/</jats:ext-link>) and Python Package (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/LondonBiofoundry/basicsynbio">https://github.com/LondonBiofoundry/basicsynbio</jats:ext-link>). With basicsynbio, users can access commonly used BASIC parts and linkers while robustly designing new parts and assemblies with exception handling for common design errors. Users can export sequence data and create build instructions for manual or acoustic liquid-handling platforms. The generation of build instructions relies on the BasicBuild Open Standard which is easily parsed for bespoke workflows and is serializable in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio by assembling a collection of 30 vectors using various sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA collection is compatible with BASIC and Golden Gate using BsaI. It encompasses vectors containing six antibiotic resistance mark
Lalvani A, Hakki S, Singanayagam A, et al., 2022, Transmissibility of SARS-CoV-2 among fully vaccinated individuals reply, LANCET INFECTIOUS DISEASES, Vol: 22, Pages: 18-19, ISSN: 1473-3099
- Author Web Link
- Citations: 1
Han P, Go MK, Chow JY, et al., 2021, A high-throughput pipeline for scalable kit-free RNA extraction, Scientific Reports, Vol: 11, Pages: 1-10, ISSN: 2045-2322
An overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications.
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 Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
- Author Web Link
- Citations: 2
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
- Author Web Link
- Citations: 17
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.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.