Imperial College London

ProfessorPaulFreemont

Faculty of MedicineDepartment of Infectious Disease

Chair in Protein Crystallography
 
 
 
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Contact

 

+44 (0)20 7594 5327p.freemont

 
 
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Location

 

259Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

280 results found

Moore SJ, Lai H-E, Chee S-M, Toh M, Coode S, Chengan K, Capel P, Corre C, de los Santos ELC, Freemont PSet al., 2021, A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology, ACS SYNTHETIC BIOLOGY, Vol: 10, Pages: 402-411, ISSN: 2161-5063

Journal article

Kelwick RJR, Webb AJ, Wang Y, Heliot A, Allan F, Emery AM, Templeton MR, Freemont PSet al., 2021, AL-PHA beads: bioplastic-based protease biosensors for global health applications, Materials Today, Vol: in press, ISSN: 1369-7021

Journal article

Wu C-H, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy-Maumus G, Freemont PS, Grundling Aet al., 2021, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Journal of Biological Chemistry, ISSN: 0021-9258

Journal article

Young R, Haines M, Storch M, Freemont PSet 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.

Journal article

Wu C-H, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy-Maumus G, Freemont PS, Gründling Aet al., 2020, Bacillus subtilis 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 g

Journal article

Moore SJ, Lai H-E, Chee S-M, Toh M, Coode S, Capel P, Corre C, de los Santos ELC, Freemont PSet al., 2020, A Streptomyces venezuelae 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>

Journal article

Crone MA, Priestman M, Ciechonska M, Jensen K, Sharp DJ, Anand A, Randell P, Storch M, Freemont PSet 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

Journal article

Crone M, Priestman M, Ciechonska M, Jensen K, Sharp D, Anand A, Randell P, Storch M, Freemont Pet 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.

Journal article

Graham N, Junghans C, Downes R, Sendall C, Lai H, McKirdy A, Elliott P, Howard R, Wingfield D, Priestman M, Ciechonska M, Cameron L, Storch M, Crone MA, Freemont PS, Randell P, McLaren R, Lang N, Ladhani S, Sanderson F, Sharp DJet 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.

Journal article

Graham NSN, Junghans C, Downes R, Sendall C, Lai H, McKirdy A, Elliott P, Howard R, Wingfield D, Priestman M, Ciechonska M, Cameron L, Storch M, Crone MA, Freemont PS, Randell P, McLaren R, Lang N, Ladhani S, Sanderson F, Sharp DJet al., 2020, SARS-CoV-2 infection, clinical features and outcome of COVID-19 in United Kingdom nursing homes, Publisher: Cold Spring Harbor Laboratory

<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

Working paper

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.

Journal article

Crone MA, Priestman M, Ciechonska M, Jensen K, Sharp D, Randell P, Storch M, Freemont Pet 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 article

Beal J, Goñi-Moreno A, Myers C, Hecht A, de Vicente MDC, Parco M, Schmidt M, Timmis K, Baldwin G, Friedrichs S, Freemont P, Kiga D, Ordozgoiti E, Rennig M, Rios L, Tanner K, de Lorenzo V, Porcar Met 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.

Journal article

Wilkinson MD, Lai H-E, Freemont PS, Baum Jet 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.

Journal article

Moore SJ, Lai H-E, Kelwick RJR, Chee SM, Bell DJ, Polizzi KM, Freemont PSet 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.

Journal article

Stach L, Morgan RM, Makhlouf L, Douangamath A, von Delft F, Zhang X, Freemont PSet 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.

Journal article

Freemont PS, Friedman JM, Beese LS, Sanderson MR, Steitz TAet al., 2020, Cocrystal structure of an editing complex of klenow fragment with dna (3'-5' exonuclease dna polymerase protein-dna interaction x-ray crystallography/metal ion catalysis), Structural Insights into Gene Expression and Protein Synthesis, Pages: 240-244, ISBN: 9789811215858

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3′ – 5′ exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3′ terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 Å away for the newly formed 3′ terminus. Since a 3′ terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.

Book chapter

Kopniczky MB, Canavan C, McClymont DW, Crone MA, Suckling L, Goetzmann B, Siciliano V, MacDonald JT, Jensen K, Freemont PSet 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.

Journal article

de Martín Garrido N, Crone MA, Ramlaul K, Simpson PA, Freemont PS, Aylett CHSet al., 2020, Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids, Molecular Microbiology, Vol: 113, Pages: 143-152, ISSN: 0950-382X

Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.

Journal article

Webb AJ, Kelwick R, Wang Y, Heliot A, Templeton MR, Freemont PSet al., 2019, AL-PHA beads: bioplastic-bsaed protease biosensors for global health, British Society for Parasitology Autumn Symposium, Belfast, UK

Conference paper

Lai H-E, Canavan C, Cameron L, Moore S, Danchenko M, Kuiken T, Sekeyová Z, Freemont PSet al., 2019, Synthetic biology and the United Nations, Trends in Biotechnology, Vol: 37, Pages: 1146-1151, ISSN: 0167-7799

Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).

Journal article

Freemont P, 2019, Synthetic biology industry - Data-driven design is creating new opportunities in biotechnology., Emerging Topics in Life Sciences, Vol: 3, Pages: 651-657, ISSN: 2397-8554

Synthetic biology is a rapidly emerging interdisciplinary research field that is primarily built upon foundational advances in molecular biology combined with engineering design. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies. This has spurned a rapid growth in start-up companies and the new synthetic biology industry is growing rapidly, with start-up companies receiving ~$6.1B investment since 2015 and a global synthetic biology market value estimated to be $14B by 2026. Many of the new start-upscan be grouped within a multi-layer ‘technology stack’. The ‘stack’ comprises a number of technology layers which together can be applied to a diversity of new biotechnology applications like consumer biotechnology products and living therapies. The ‘stack’ also enables new commercial opportunities and value chains similar to the software design and manufacturing revolution of the 20th century. However, synthetic biology industry is at a crucial point, as it now requires recognisable commercial successes in order for the industry to expand and scale, in terms of investment and companies. However, such expansion may directly challenge the ethos of synthetic biology, in terms of open technology sharing and democratisation, which could by accident lead to multi-national corporations and technology monopolies similar to the existing biotechnology/biopharma industry.

Journal article

Wood TE, Howard SA, Forster A, Nolan LM, Manoli E, Bullen NP, Yau HCL, Hachani A, Hayward RD, Whitney JC, Vollmer W, Freemont PS, Filloux Aet al., 2019, The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology, Cell Reports, Vol: 29, Pages: 187-201.e7, ISSN: 2211-1247

The type VI secretion system (T6SS) is crucialin interbacterial competition and is avirulence determinant ofmany Gram-negative bacteria. Several T6SS effectorsarecovalently fused to secreted T6SS structural components such asthe VgrG spike for delivery into target cells.In Pseudomonas aeruginosa, theVgrG2b effector waspreviously proposedto mediatebacterial internalisation into eukaryotic cells. In this work, wefind that the VgrG2b C-terminal domain(VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein.We resolve thestructure of VgrG2bC-ter and confirm it is a member ofthezinc-metallopeptidasefamily of enzymes. We show that this effector causesmembrane blebbing atmidcell, whichsuggests a distincttype of T6SS-mediated growthinhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Ourstudyintroduces a further effector family to the T6SS arsenaland demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.

Journal article

Kelwick RJR, Ricci L, Chee SM, Bell D, Webb A, Freemont Pet 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.

Journal article

Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Le Feuvre R, Friedman DC, Fu X, Gold ND, Herrgard MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, Freemont PSet al., 2019, Building a global alliance of biofoundries (vol 10, 2040, 2019), Nature Communications, Vol: 10, Pages: 1-2, ISSN: 2041-1723

Journal article

Rajakumar PD, Gower G, Suckling L, Kitney R, McClymont D, Freemont Pet al., 2019, Rapid prototyping platform for Saccharomyces cerevisiae using computer-aided genetic design enabled by parallel software and workcell platform development, Slas Technology, Vol: 24, Pages: 291-297, ISSN: 2472-6303

Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

Journal article

Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Le Feuvre R, Friedman DC, Fu X, Gold ND, Herrgard MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, Freemont PSet al., 2019, Building a global alliance of biofoundries, NATURE COMMUNICATIONS, Vol: 10, Pages: 1-4, ISSN: 2041-1723

Journal article

Kelwick R, Webb AJ, Wang Y, Allan F, Freemont Pet al., 2019, ISEV2019 Abstract Book. PT09.10: Protease biomarker detection using functionalised bioplastic-based biosensors, ISEV 2019, Publisher: Co-Action Publishing, ISSN: 2001-3078

Conference paper

Thaore V, Moore S, Polizzi K, Freemont P, Shah N, Kontoravdi Cet al., 2019, Cell-free multi-enzyme system for the industrial production of fine chemicals, Chemical Engineering Day UK 2019

Conference paper

Suckling L, McFarlane C, Sawyer C, Chambers SP, Kitney RI, McClymont DW, Freemont PSet al., 2019, Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation, Synthetic and Systems Biotechnology, Vol: 4, Pages: 57-66, ISSN: 2405-805X

High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.

Journal article

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