Results
- Showing results for:
- Reset all filters
Search results
-
Journal articleCrone 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>
-
Journal articleBeal 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: 1469-221XSynthetic 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.
-
Journal articleWilkinson 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: 0066-4804Advances 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.
-
Journal articleMoore 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: 2161-5063It 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.
-
Journal articleWilliams R, Yates L, Zhang X, 2020,
Structures and regulations of ATM and ATR, master kinases in genome integrity
, Current Opinion in Structural Biology, Vol: 61, Pages: 98-105, ISSN: 0959-440XHomologous recombination (HR) is a faithful repair mechanism for double stranded DNA breaks. Two highly homologous master kinases, the tumour suppressors ATM and ATR (Tel1 and Mec1 in yeast), coordinate cell cycle progression with repair during HR. Despite their importance, our molecular understanding of these apical coordinators has been limited, in part due to their large sizes. With the recent development in cryo-electron microscopy, significant advances have been made in structural characterisation of these proteins in the last two years. These structures, combined with new biochemical studies, now provide a more detailed understanding of how a low basal activity is maintained and how activation may occur. In this review, we summarize recent advances in the structural and molecular understanding of these key components in HR, compare the common and distinct features of these kinases and suggest aspects of structural components that are likely to be involved in regulating its activity.
-
Journal articleWigley DB, Willhoft O, 2020,
INO80 and SWR1 complexes: the non-identical twins of chromatin remodelling
, Current Opinion in Structural Biology, Vol: 61, Pages: 50-58, ISSN: 0959-440XThe INO80 family of chromatin remodellers are multisubunit complexes that perform a variety of tasks on nucleosomes. Family members are built around a heterohexamer of RuvB-like protein, an ATP-dependent DNA translocase,nuclear actin and actin-related proteins, and a few complex-specific subunits. They modify chromatin in a number of ways including nucleosome sliding and exchange of variant histones. Recent structural information on INO80 and SWR1 complexes has revealed similarities in the basic architecture of the complexes. However, structural and biochemical data on the complexes bound to nucleosomes reveal these similarities to be somewhat superficial and their biochemical activities and mechanisms are very different. Consequently, the INO80 family displays a surprising diversity of function that is based upon a similar structural framework.
-
Journal articleZhang X, Blundell TL, 2020,
Editorial overview: Macromolecular assemblies
, CURRENT OPINION IN STRUCTURAL BIOLOGY, Vol: 61, Pages: VI-VIII, ISSN: 0959-440X -
Journal articleStach 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: 0014-5793Several 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.
-
Journal articleGao F, Danson AE, Ye F, et al., 2020,
Bacterial enhancer binding proteins - AAA+ proteins in transcription activation
, Biomolecules, Vol: 10, ISSN: 2218-273XBacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.
-
Journal articleYates L, Williams R, Hailemariam S, et al., 2020,
Cryo-EM structure of nucleotide-bound Tel1ATM unravels the molecular basis of inhibition and structural rationale for disease-associated mutations
, Structure, Vol: 28, Pages: 96-104.e3, ISSN: 0969-2126Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases centralto the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia(A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Usingcryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Ourstructure reveals molecular details of key residues surrounding the nucleotide binding site andprovides a structural and molecular basis for its intrinsically low basal activity. We show thatthe catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatorydomain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an openconformation, thus explaining the requirement for Tel1 activation. Structural comparisons withother PIKKs suggest a conserved and common allosteric activation mechanism. Our work alsoprovides a structural rationale for many mutations found in A-T and cancer.
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.
General enquiries
Section Manager
Brett Onslow
+44 (0)20 7594 3871
Personal Assistant for the Section of Structural Biology
Kasia Pearce
+44 (0)20 7594 2917
Laboratory Manager
Soo Mei Chee