Establishing a Robotic

Understanding the transcriptional regulationnetworks that control the gene expression programs in a living cell is one of fundamental questions in molecular biology. Using single perturbations allows us to identify the key players in those networks. To better understand the structure, we can use double perturbations to measure genetic interactions between the components in the network. By systematically analyzing all the genetic interactions between the genes in the network we can reconstruct it and to characterize the relations between its components. However, such a systematic approach is challenging because the number of pair-wise interactions grows at the square of the number of genes. Thus, a high-throughput measurement system is needed. In this work I describe the establishment of a robotic high throughput measurement system for dissection of transcriptional networks in the budding yeast. We adapted a novel robotic high throughput system for manipulating yeast strains in multi-well microtiter plates (in either 96 or 384 well format). We developed and calibrated a robotic growth protocol that allows us to grow the strains on such plates under optimal and homogeneous growth conditions and brings them to a predetermined density range at the beginning of the experiment. The cells are exposed to arrays of stresses, and then the transcriptional response of the entire plate is monitored in a single cell level by an automated flow cytometry measurement system. We developed an algorithm for partitioning events from the flow cytometry data stream of the whole plate to their correct wells. We used the system to perform a systematic inspection of the budding yeast general stress response network. A generic response to stress that is mediated by two transcription factors, Msn2 and Msn4, which regulate the transcription of hundreds of genes. We identified the main genes playing a role in this network and evaluated the effect of single and double perturbations in the network on Msn2/4 activity in response to several stresses.