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Synthetic biology in yeast : reconstructing the galactose network to probe the role of feedback induction in response to metabolic stimuli

  • Author(s): Ferry, Michael Stephen
  • et al.
Abstract

With the expanding interest in cellular responses to dynamic environments, microfluidic devices have become important experimental platforms for biological research. Microfluidic "microchemostat" devices enable precise environmental control while capturing high quality, single cell gene expression data. For studies of population heterogeneity and gene expression noise, these abilities are crucial. I have developed a microchemostat device optimized for capturing data from thousands of cells in multiple sub-experiments. The device is robust, easy to use and capable of generating precisely controlled dynamic environments. The device uses an integrated fluidic junction, coupled to linear actuators, to modulate the external port pressures as a function of time. In this way the concentration of an inducer compound can be tightly controlled without the use of mechanical mixing devices. To analyze the large amounts of data generated, I have developed a method for automated cell tracking, focusing on the special problems presented by Saccharomyces cerveisiae cells. I have used these tools to probe the response of a natural genetic circuit, the Gal system of S. cerevisiae. I have altered the regulation of the native Gal system, replacing the transcriptional positive and negative feedback loops with artificial promoters. Looking at these modified strains, I have determined that induced negative feedback is essential for tuning the cell's genetic response to the external galactose concentration. Moreover, without induced negative feedback, the system exhibits bistability, with subpopulations of responding and non-responding cells. When observing these cells in a dynamic environment, I have found that the Gal network is optimized to respond sharply to changes in the inducer concentration, regardless of the network's original induction state

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