Effects of Transcriptional and Post-Transcriptional Control Mechanisms on Biological Noise in Synthetic Gene Circuits

Synthetic Biology is an interdisciplinary research field seeking to correct faulty cellular processes or implement predictable de-novo tasks by engineering biological systems. In this perspective, the potential of developing biosynthetic devices of industrial and medical relevance is hindered by the requirement of accounting for, controlling and finally exploiting the randomness of biochemical events through which biological complexity is implemented. In this thesis mathematical modelling and experimental acquisitions of basic synthetic circuits are adopted to guide the selection of gene expression control mechanisms and network topologies in the design of synthetic devices able to reliably operate in the stochastic cellular context. To this end, a noise tester circuits’ catalogue, intended as a tool for quantitatively investigating the robustness of newly designed synthetic devices, is implemented. Two synthetic gene circuits, exerting either a transcriptional or post-transcriptional control in the expression of a fluorescent reporter, are selected from the circuits’ library for detailed characterization. Based on bulk measurements, deterministic models are defined to identify the kinetic rates of biochemical reactions governing the circuits’ function. The inherently derived stochastic models are further used in numerical computations of plasmid copy number effect on gene expression stochasticity. Subsequently, flow cytometry analysis is used to quantify the steady-state dispersion in protein levels within an isogenic population of transformants. An intriguing feature of the stochastic models describing the observed variance in protein levels is the necessity of including extrinsic components (e.g. cell division events). Numerical analysis identified post-transcriptional control as the best candidate for noise minimization. Finally, we report the results of research undertaken during a period staying at the “Centre for Synthetic and System Biology” of the University of Edinburgh, where the phenotypic consequences of a long-non coding RNA on the transcriptional activation of GAL1-10 promoter in Saccharomyces Cerevisiae are investigated using fluorescence microscopy and microfluidics.