Hon Ming Yip
Cells consume extra-cellular nutrients and resources to maintain cellular fitness. Extra-cellular conditions vary over time. Cellular programs encoded in genes adjust to adapt to the environments. Gene regulatory networks (GRNs) have evolved to be responsible for this task through regulation of a set of genes. The gene products work collectively, and assist cells to optimise their survival rate under nutrient limiting conditions. The PHO system is an example. It is a phosphate-responsive pathway found in S. cerevisiae. PHO genes are turned on when intra-cellular phosphate levels are limiting. The functions of individual genes in the PHO system are rather well-studied. However, the program that governs these genes at the system level is not yet fully understood. Systematic characterisation requires measurements of a large number of strains and environmental conditions. Conducting experiments while controlling consumable nutrients is especially challenging because the nutrient level decreases over time in a closed system. Chemostats allow stablilisation of nutrient levels, but have a lack of throughput. Conventional measurement instruments such as plate readers and cytometers do not satisfy experimental requirements including sub-cellular measurement, stable nutrient levels, and high throughput.Microfluidics is an experimental tool that is able to solve these bottlenecks. In this work, two microfluidic platforms were developed to satisfy the aforementioned requirements. With time-lapse fluorescence imaging, 1) single cell level measurement, 2) high strain and media condition throughput, 3) continuous media supply, and 4) temporally changing input control can be achieved by the proposed platforms. This work finely mapped the system response as a function of inorganic phosphate level. The sub-cellular localisation of the master transcription factor Pho4 and the PHO promoters' activities were studied at the single cell level under high resolution and well-controlled media conditions. Four program states were identified in the GRN and two promoter clusters were found. A new localisation state was also found in Pho4. These results resolved an outstanding question of how this system is programmed.
EPFL2022
