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Rapid Pathogen Detection Using Synthetic Biology

Principal Supervisor: Professor Tim Dafforn - School of Biosciences

Co-supervisor: Jim Tucker (Industrial input from SASA, Scotland who have offered financial support)

PhD project title: Rapid Pathogen Detection Using Synthetic Biology

University of Registration: Birmingham

Project outline:

Bacterial contamination has significant influence on our food security. On one hand, contamination of food with bacteria that are human pathogens represents a significant risk to human health. On the other hand, bacterial contamination can lead to degradation of food before it even reaches the shop and indeed even once in the shop, it is an important determinant of shelf life.

Two examples show the scale of this type of bacterial contamination issue in food production:

  1. In 2011 an outbreak of E. coli O102 occurred in central Europe which hospitalised a large number of people and eventually killed 57. Unreliable detection methods led to the belief that the infection came from Spanish cucumbers which cost the Spanish cucumber industry more than $200 million per week in lost revenue. Eventually the outbreak was traced to fenugreek seeds from the Middle East. The presence of a rapid and reliable detection system could have reduced the spread of the infection more quickly and hence saved lives and the cost to the cucumber industry.
  2. Bacterial contamination of bulk stored food crops, intended for human consumption, is a worldwide problem. Tesco Market research indicates that the typical pack-house handling 73,000 tonnes of potatoes loses approximately £155k annually to soft rot in potatoes alone. Rapid detection of the bacteria that cause soft rot would allow packing houses to detect the issue early allowing processing systems to be decontaminated.

Central to mitigating both of these problems is a need to rapidly detect both forms of contamination in a simple and cost effective test in order to initiate decontamination measures.

In this project we aim to continue our development of a rapid assay system based on M13 Bacteriophage and Linear Dichroism (LD) spectroscopy. Our assay is based on the observation that long molecules, like M13 bacteriophage, align in fluid flow. When aligned, chromophores within the molecule have an LD signal . Over the past 5 years our laboratories have shown that M13 bacteriophage can be engineered to bind specifically to pathogens, such as E. coli O157. In our first experiments this was achieved by simply adding antibodies to the surface of the M13. These M13-conjugates bind to antigens on the surface of the pathogen, disrupting of the alignment of M13 and hence the LD signal. In later experiments we extended this approach by further modifying the M13 scaffold, allowing it to detect a range of other targets including small molecules (e.g. explosives and drugs of abuse) and DNA.

This has now provided the exciting opportunity to carry out a multimodal test (one that detects surface antigens, DNA and small molecules produced by a target simultaneously) for the first time.

In this project we aim to develop such a multimodal test to detect food spoilage organisms and food-borne pathogens. For the former case we will investigate the potential to detect of pectolytic bacteria, while in the latter we will continue to develop our detection system for E. coli O157 and Salmonella.

The outcome of the project will be:

  1. An understanding of the challenges present in the development of a multimodal assay system based on LD. This will include a complete study of experimental parameters (e.g. M13-antibody conjugation levels, non-specific binding and reagent compatibility).
  2. Development of an LD multimodal detection protocol
  3. At least 2 prototype assays.

References:

  1. Carr-Smith J., Pacheco-Gómez R., Little H.A., Hicks M.R., Sandhu S., Steinke N., Smith D.J., Rodger A., Goodchild S.A., Lukaszewski R.A., Tucker J.H.R, Dafforn T.R. “Polymerase Chain Reaction on a Viral Nanoparticle.” ACS Synth. Biol. 2015, 4, 1316-1325.
  2. Daniela P. Lobo, Alan M. Wemyss, David J. Smith, Anne Straube, Kai B. Betteridge, Andrew H.J. Salmon, Rebecca R. Foster, Hesham E. Elhegni, Simon C. Satchell, Haydn A. Little, Raúl Pacheco-Gómez, Mark J. Simmons, Matthew R. Hicks, David O. Bates, Alison Rodger, Timothy R. Dafforn, and Kenton P. Arkill, (2015) Direct detection and measurement of wall shear stress using a filamentous bio-nanoparticle. Nano Research 2015, 8(10):3307–3315
  3. Pacheco-Gomez, R., J. Kraemer, S. Stokoe, H.J. England, C.W. Penn, E. Stanley, A. Rodger, J. Ward, M.R. Hicks, and T.R. Dafforn, “Detection of Pathogenic Bacteria Using a Homogeneous Immunoassay Based on Shear Alignment of Virus Particles and Linear Dichroism.” Analytical Chemistry 2012. 84(1): p. 91-97.

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

  • Protein Expression and Purification
  • Bioconjugation chemistry
  • Bacteriophage M13 engineering
  • Linear Dichroism Spectroscopy (including instrument development)

Contact: Professor Tim Dafforn, University of Birmingham