Professor Tim Overton

Can we eliminate biofilms by feeding bacteria differently?

Secondary Supervisor(s): Prof Jessica Blair

University of Registration: University of Birmingham

BBSRC Research Themes:

• Understanding the Rules of Life (Microbiology)

Project Outline

Most bacteria in nature live in biofilms, communities that are usually attached to solid surfaces and protected by a self-produced matrix of polymer molecules. Biofilms are frequently highly resistant to antibiotics, and are very sticky so difficult to remove from surfaces. Biofilms represent a massive problem for humanity, causing ~80% of infections, fouling pipes, and increasing fuel usage on ships. The economic impact of biofilms has recently been estimated to be ~ US$4000 billion per year.

We have recently discovered a fundamental link between energy metabolism and biofilm formation. This links to a body of literature suggesting that respiration can alter biofilm development and, more broadly, to studies on how energy metabolism affects bacterial physiology and behaviour. This project will explore these links to understand how biofilms can be prevented and removed by modulating energy metabolism.

Our main research questions are:

• How is the energy state of the cell linked to biofilm formation?
• How is this link regulated? What are the sensors and regulators involved in this process?
• How can we use this link to prevent biofilm formation, and remove biofilms that have already formed?

We will use a combination of approaches, grounded in single-cell analysis of bacteria using flow cytometry, confocal microscopy, and molecular biology. This will allow us to measure how individual bacteria respond to alterations in their energy state, and how they change regulation of genes responsible for aspects of biofilm formation (such as adhesins and matrix polysaccharides). We will start work in E. coli and Salmonella but expect to expand into other species to test the broader implications of the link between energy and biofilms.

This interdisciplinary project is supervised by Professor Tim Overton, an expert on biofilms, single-cell analysis of bacteria, and bacterial physiology, and Professor Jessica Blair, an expert on bacterial efflux pumps and antimicrobial resistance.

How can we direct recombinant proteins into the periplasm efficiently?

Secondary Supervisor(s): Professor Owen Thomas

University of Registration: University of Birmingham

BBSRC Research Themes:

• Renewable Resources and Clean Growth (Industrial Biotechnology)

Project Outline

Recombinant protein production is a major technique used in biotechnology where a host organism is used to synthesise proteins such as drugs, enzymes, or for structural studies. The host we use is the bacterium E. coli, which is good at making relatively simple proteins such as insulin or antibody fragments. Although E. coli cannot perform as many post-translational modifications as mammalian cells, it can form disulphide bonds, which are important for the folding and function of many products.

To form disulphide bonds, the recombinant protein is usually directed to the periplasm (the space between the inner and outer membranes) where the enzymes that create disulphide bonds are located. This means that the protein must pass through the inner membrane, a step which makes processes more complicated and can cause issues with protein misfolding and cell death. We would like to improve this process.

In this project we will explore how we can best improve translocation of recombinant proteins into the periplasm. We have previously developed optimised signal peptides, fermentation processes to improve periplasmic production, and feedback systems to allow us to sense when bacteria are stressed and allow production to be automatically modulated. In this project we will explore these approaches to improve the quantity and quality of accumulated periplasmic protein and de-risk processes to ensure success.

We will use a combination of approaches, grounded in single-cell analysis using flow cytometry, fermentation development, and molecular biology.

This interdisciplinary project will be supervised by Professor Tim Overton, an expert on bioprocess development, single-cell analysis of bacteria, and bacterial physiology, and Professor Owen Thomas, an expert in downstream processing of proteins.