Understanding the coordination between cell growth, volume and division and DNA replication

Optical density (OD) is a widely used technique of monitoring growth of bacterial cells in a suspension. OD is given as a logarithmic ratio between light falling onto the bacterial cells in a solution and the light transmitted through the solution. The wavelength of light most widely used for OD measurements is 600nm and measuring it with a spectrometer at different time points of bacterial culture growth results in characteristic 'growth curves'. However, the information on volume, shape and specific characteristics (such as chromosome shape, internal pH levels) of cells within the population are not accessible through growth curves. Yet, this information is of interest. Cells change during growth in liquid culture; be that when grown in different media, treated with antibiotics, or as they are producing recombinant proteins for biotechnology purposes. To fully understand these relationships and therefore bacterial growth, we need to be able to measure it on a single cell level, with high-throughput. Understanding bacterial growth is not only an important basic-science question, but also has wide applicability. For example, the capability to monitor individual bacterial cells during production is a technical innovation that can transform assessment of compound production within biotechnology industry. Key enzymes in metabolic pathways for compound production can be fluorescently labelled to identify their presence at required branch point during production. Changes in internal pH and ATP can be monitored simultaneously to determine which metabolic pathways have detrimental effects on the cell and cause production failure. Similarly, the complexity of molecular interactions initiated by antibiotic binding the target, make it extraordinary difficult to elucidate the underlying mechanism(s) of cell lysis. It is not surprising that although the targets of antibiotic attack are often known, the mechanisms of killing remain concealed. Monitoring changes in cellular physiology, in each cell and during antibiotic treatment, is a good way of tackling the complexity. Cellular physiology integrates the molecular mechanisms and helps identify functionally most relevant molecular processes that lead to cell death.

With this in mind, the PhD project will move current state-of-the-art technology for monitoring bacterial growth to enable us to assess individual cells during growth in a population. Using a microfluidic and microscopy based platform the student will develop high-throughput imaging of individual bacterial cells within the population, while simultaneously measuring 'growth curves'. The platform will be used to fully characterize and subsequently mathematically model, the relationship between bacterial volume, chromosome shape and division and the bacterial growth rates. At the beginning, the student will look at the media of different 'richness', but later in the project effects of adding different antibiotics will be studied. Monitoring cell volume, pH levels, ATP and chromosome shape, as the cells are exposed to different antibiotics, will enable us to better understanding antibiotic killing mechanisms.