Imperial College London

ProfessorPaulFreemont

Faculty of MedicineDepartment of Medicine

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

246 results found

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, ISSN: 2041-1723

JOURNAL ARTICLE

Thaore V, Moore S, Polizzi K, Freemont P, Shah N, Kontoravdi Cet al., 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

Kylilis N, Riangrungroj P, Lai H-E, Salema V, Fernández LÁ, Stan G-BV, Freemont PS, Polizzi KMet al., 2019, Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications., ACS Sens, Vol: 4, Pages: 370-378

Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine.

JOURNAL ARTICLE

Kylilis N, Riangrungroj P, Lai H-E, Salema V, Angel Fernandez L, Stan G-BV, Freemont PS, Polizzi KMet al., 2019, Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications, ACS SENSORS, Vol: 4, Pages: 370-378, ISSN: 2379-3694

JOURNAL ARTICLE

Exley K, Reynolds CR, Suckling L, Chee SM, Tsipa A, Freemont PS, McClymont D, Kitney RIet al., 2019, Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway, JOURNAL OF BIOLOGICAL ENGINEERING, Vol: 13, ISSN: 1754-1611

JOURNAL ARTICLE

Tosi T, Hoshiga F, Millership C, Singh R, Eldrid C, Patin D, Mengin-Lecreulx D, Thalassinos K, Freemont P, Grundling Aet al., 2019, Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM, PLOS PATHOGENS, Vol: 15, ISSN: 1553-7366

JOURNAL ARTICLE

Silhan J, Zhao Q, Boura E, Thomson H, Forster A, Tang CM, Freemont PS, Baldwin GSet al., 2018, Structural basis for recognition and repair of the 3 '-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis, NUCLEIC ACIDS RESEARCH, Vol: 46, Pages: 11980-11989, ISSN: 0305-1048

JOURNAL ARTICLE

Thaore V, Moore S, Polizzi K, Freemont P, Shah N, Kontoravdi Cet al., Techno-economic evaluation of a cell-free syntheticbiochemistry route for raspberry ketone production atindustrial scale, Vaishali Thaore

CONFERENCE PAPER

Rajakumar PD, Gowers G-OF, Suckling L, Foster A, Ellis T, Kitney RI, McClymont DW, Freemont PSet al., 2018, Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development., SLAS Technol

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

Moore SJ, MacDonald JT, Wienecke S, Ishwarbhai A, Tsipa A, Aw R, Kylilis N, Bell DJ, McClymont DW, Jensen K, Polizzi KM, Biedendieck R, Freemont PSet al., 2018, Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 115, Pages: E4340-E4349, ISSN: 0027-8424

JOURNAL ARTICLE

Salih O, He S, Planamente S, Stach L, MacDonald JT, Manoli E, Scheres SHW, Filloux A, Freemont PSet al., 2018, Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa, STRUCTURE, Vol: 26, Pages: 329-+, ISSN: 0969-2126

JOURNAL ARTICLE

Hazel P, Kroll SHB, Bondke A, Barbazanges M, Patel H, Fuchter MJ, Coombes RC, Ali S, Barrett AGM, Freemont PSet al., 2018, Inhibitor Selectivity for Cyclin-Dependent Kinase7: A Structural, Thermodynamic, and Modelling Study (vol 12, pg 372, 2017), CHEMMEDCHEM, Vol: 13, Pages: 207-207, ISSN: 1860-7179

JOURNAL ARTICLE

Webb A, Allan F, Kelwick R, Jensen K, Templeton M, Freemont Pet al., 2018, PROTEASE-BASED BIOREPORTERS FOR THE DETECTION OF <it>SCHISTOSOMA CERCARIAE</it>, 67th Annual Meeting of the American-Society-of-Tropical-Medicine-and-Hygiene (ASTHM), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 191-191, ISSN: 0002-9637

CONFERENCE PAPER

Kelwick R, Ricci L, Chee SM, Bell D, Webb AJ, Freemont PSet al., 2018, Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics, Synthetic Biology, Vol: 3

JOURNAL ARTICLE

Lai H-E, Moore S, Polizzi K, Freemont Pet al., 2018, EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology., Pages: 429-444

Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.

BOOK CHAPTER

Lai H-E, Obled AMC, Chee SM, Morgan R, Sharma S, Moore S, Polizzi K, Goss RJM, Freemont Pet al., 2017, A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Integrating synthetic chemistry with synthetic biology allows rapid access to xenobiotic compounds which may provide improved therapeutic activity. By supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with eight tryptophan substrate analogues or tryptophan halogenase RebH in vivo , 68 new-to-nature analogues of violacein were generated. Furthermore, 20 new derivatives were generated from brominated analogues via Suzuki-Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein, we demonstrate a flexible and rapid approach to access diverse chemical space that can be applied to a wide range of natural product scaffolds.

THESIS DISSERTATION

Wen KY, Cameron L, Chappell J, Jensen K, Bell DJ, Kelwick R, Kopniczky M, Davies JC, Filloux A, Freemont PSet al., 2017, A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples., ACS Synthetic Biology, Vol: 6, Pages: 2293-2301, ISSN: 2161-5063

Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.

JOURNAL ARTICLE

Stach L, Freemont PS, 2017, The AAA+ ATPase p97, a cellular multitool, BIOCHEMICAL JOURNAL, Vol: 474, Pages: 2953-2976, ISSN: 0264-6021

JOURNAL ARTICLE

Smith WD, Bardin E, Cameron L, Edmondson CL, Farrant KV, Martin I, Murphy RA, Soren O, Turnbull AR, Wierre-Gore N, Alton EW, Bundy JG, Bush A, Connett GJ, Faust SN, Filloux A, Freemont PS, Jones AL, Takats Z, Webb JS, Williams HD, Davies JCet al., 2017, Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis, FEMS MICROBIOLOGY LETTERS, Vol: 364, ISSN: 0378-1097

JOURNAL ARTICLE

Moore SJ, MacDonald JT, Freemont PS, 2017, Cell-free synthetic biology for in vitro prototype engineering, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 45, Pages: 785-791, ISSN: 0300-5127

JOURNAL ARTICLE

Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KMet al., 2017, Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production, BIOTECHNOLOGY AND BIOENGINEERING, Vol: 114, Pages: 1290-1300, ISSN: 0006-3592

JOURNAL ARTICLE

Moore SJ, Lai H-E, Needham H, Polizzi KM, Freemont PSet al., 2017, Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool, BIOTECHNOLOGY JOURNAL, Vol: 12, ISSN: 1860-6768

JOURNAL ARTICLE

McClymont DW, Freemont PS, 2017, With all due respect to Maholo, lab automation isn't anthropomorphic, NATURE BIOTECHNOLOGY, Vol: 35, Pages: 312-314, ISSN: 1087-0156

JOURNAL ARTICLE

Hazel P, Kroll SHB, Bondke A, Barbazanges M, Patel H, Fuchter MJ, Coombes RC, Ali S, Barrett AGM, Freemont PSet al., 2017, Inhibitor Selectivity for Cyclin-Dependent Kinase7: AStructural, Thermodynamic, and Modelling Study, CHEMMEDCHEM, Vol: 12, Pages: 372-380, ISSN: 1860-7179

JOURNAL ARTICLE

Webb AJ, Kelwick R, Freemont PS, 2017, Opportunities for applying whole-cell bioreporters towards parasite detection, MICROBIAL BIOTECHNOLOGY, Vol: 10, Pages: 244-249, ISSN: 1751-7915

JOURNAL ARTICLE

Freemont P, 2017, Synthesising, Biologist, Vol: 64, Pages: 22-25, ISSN: 0006-3347

JOURNAL ARTICLE

Kelwick R, Webb AJ, MacDonald JT, Freemont PSet al., 2016, Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements, METABOLIC ENGINEERING, Vol: 38, Pages: 370-381, ISSN: 1096-7176

JOURNAL ARTICLE

MacDonald JT, Freemont PS, 2016, Computational protein design with backbone plasticity, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 44, Pages: 1523-1529, ISSN: 0300-5127

JOURNAL ARTICLE

Moore SJ, Lai H-E, Kelwick RJR, Ghee SM, Bell DJ, Polizzi KM, Freemont PSet al., 2016, EcoFlex: A Multifunctional MoClo Kit for E-coli Synthetic Biology, ACS SYNTHETIC BIOLOGY, Vol: 5, Pages: 1059-1069, ISSN: 2161-5063

JOURNAL ARTICLE

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