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

237 results found

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

Kylilis N, Riangrungroj P, Lai H-E, Salema V, Fernandez LA, Stan G-B, Freemont P, Polizzi Ket al., 2018, A low-cost biological agglutination assay for medical diagnostic applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of 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 supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the 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 both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.

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

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

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

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

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

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

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

Lai H-E, Chee SM, Morgan M, Moore S, Polizzi K, Freemont Pet al., 2017, A semi-synthetic strategy for derivatization of the violacein natural product scaffold

The next frontier in drug discovery could be the semi-synthesis of non-natural, xenobiotic compounds combining both natural product biosynthesis and synthetic chemistry. However, the required tools and underlying engineering principles are yet to be fully understood. One way to investigate non-natural product biosynthesis is to probe the substrate promiscuity of a clinically relevant biosynthesis pathway. Violacein is a bisindole compound produced by the VioABCDE biosynthesis pathway using L-tryptophan as the starting substrate. Previous studies have shown that violacein exhibits antimicrobial properties, and synthetic analogues of violacein might give rise to new targets for therapeutic development to combat antimicrobial resistance. By adding seven types of tryptophan analogues available commercially, 62 new violacein or deoxyviolacein analogues were generated with a synthetic violacein biosynthesis pathway expressed in Escherichia coli , demonstrating the promiscuity of violacein biosynthesis enzymes. Growth inhibition assays against Bacillus subtilis , a Gram-positive bacterium, were carried out to measure growth inhibitory activity of violacein analogues compared to violacein. In addition, we show that four new 7-chloro analogues of violacein or deoxyviolacein can be generated in vivo by combining the rebeccamycin and violacein biosynthesis pathways and purified 7-chloro violacein was found to have similar growth inhibitory activity compared to violacein. Structural studies of VioA revealed active site residues that are important for catalytic activity, and further pathway recombination with VioA homologues in related bisindole pathways may lead to more efficient enzymes that would accept tryptophan analogues more readily.

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

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

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

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

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

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

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

Chambers S, Kitney R, Freemont P, 2016, The Foundry: the DNA synthesis and construction Foundry at Imperial College, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 44, Pages: 687-688, ISSN: 0300-5127

JOURNAL ARTICLE

Filloux A, Freemont P, 2016, Baseplates in contractile machines, NATURE MICROBIOLOGY, Vol: 1

JOURNAL ARTICLE

Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, Garcia LDA, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis Tet al., 2016, Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 113, Pages: E3431-E3440, ISSN: 0027-8424

JOURNAL ARTICLE

Florea M, Reeve B, Abbott J, Freemont PS, Ellis Tet al., 2016, Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582, SCIENTIFIC REPORTS, Vol: 6, ISSN: 2045-2322

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

Kelwick R, Webb AJ, Macdonald JT, Freemont PSet al., 2016, Development of a bacillus subtilis cell-free transcriptiontranslation system

CONFERENCE PAPER

Kopniczky M, Jensen K, Freemont P, 2016, Introducing the human cell-free TX-TL system as a new prototyping platform for mammalian synthetic biology

CONFERENCE PAPER

Lund-Palau H, Turnbull AR, Bush A, Bardin E, Cameron L, Soren O, Wierre-Gore N, Alton EWFW, Bundy JG, Connett G, Faust SN, Filloux A, Freemont P, Jones A, Khoo V, Morales S, Murphy R, Pabary R, Simbo A, Schelenz S, Takats Z, Webb J, Williams HD, Davies JCet al., 2016, Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches, EXPERT REVIEW OF RESPIRATORY MEDICINE, Vol: 10, Pages: 685-697, ISSN: 1747-6348

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

MacDonald JT, Kabasakal BV, Godding D, Kraatz S, Henderson L, Barber J, Freemont PS, Murray JWet al., 2016, Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 113, Pages: 10346-10351, ISSN: 0027-8424

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

Planamente S, Salih O, Manoli E, Albesa-Jove D, Freemont PS, Filloux Aet al., 2016, TssA forms a gp6-like ring attached to the type VI secretion sheath, EMBO JOURNAL, Vol: 35, Pages: 1613-1627, ISSN: 0261-4189

JOURNAL ARTICLE

Polizzi KM, Freemont PS, 2016, Synthetic biology biosensors for healthcare and industrial biotechnology applications

CONFERENCE PAPER

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