Multicelled Behaviour in E. coli: Design and experimental characterization of an engineered library of hybrid promoters.

Synthetic biology has recently had a great development, many papers have been published and many applications have been presented, spanning from the production of biopharmacheuticals to the synthesis of bioenergetic substrates or industrial catalysts. But, despite these advances, most of the applications are quite simple and don’t fully exploit the potential of this discipline. This limitation in complexity has many causes, like the incomplete characterization of some components, or the intrinsic variability of the biological systems, but one of the most important reasons is the incapability of the cell to sustain the additional metabolic burden introduced by a complex circuit. The objective of the project, of which this work is part, is trying to solve this problem through the engineering of a multicellular behaviour in prokaryotic cells. This system will introduce a cooperative behaviour that will allow to implement complex functionalities, that can’t be obtained with a single cell. In particular the goal is to implement the Leader Election, this procedure has been firstly devised in the field of distributed computing, to identify the process that allow to identify a single process as organizer and coordinator of a series of tasks assigned to the whole population. The election of the Leader greatly simplifies the computation providing a centralized control. Further- more this system may even be useful to evolutionary studies that aims to explain how complex organisms evolved from unicellular systems. The work presented here describes, in particular, the design and the experimental characterization of a component of the circuit that solves the Leader Election problem. This module, composed of an hybrid promoter and a gene, is activated in the non-leader cells after receiving the signal that a leader is present in the colony. The most important element, in this case, is the hybrid promoter, it has been realized in different versions, applying the heuristic rules stated in [22], and their activity has been experimentally tested. The objective of the experimental characterization was to test the response of the genetic circuit to the introduction, in the cellular environment, of particular molecules, inducers, that can be considered inputs of the system. The desired behaviour is similar to the one of a logic AND gate in which the exit, represented by the luminous signal produced by a fluorescent protein, is one only in presence of both inducers. The robustness and the stability of this behaviour have been tested by changing the concentration of the input signals and building dose response curves. From these data it is possible to conclude that the analysed constructs have an AND-like behaviour over a wide range of inducers’ concentrations, even if it is possible to identify many differences in the expression profiles of the different constructs. This variability accounts for the fact that the input and the output signals are continuous, and so their binary representation isn’t able to capture the complexity of the behaviour. The module of the circuit that has been considered in this analysis has a fundamental role in the realization of the intercellular communication system that is necessary for the cooperative behaviour to take place. For this reason, the second phase of the characterization has been focused on the analysis of the signal transmission. In particular, the interaction between this element and the one that is responsible for emitting the chemical signal has been tested. The desired behaviour is still similar to a logic AND, since, even in this case, the exit signal is determined by the hybrid promoter activity. The experimental results have demonstrated that the systems behave correctly, even if there is still a substantial variability between them. The dose response curves highlighted that stricter constrains on the inducers concentrations need to be imposed in order to obtain a clear separation between the two levels of expression. In the conclusive chapter the DNA sequences of the hybrid promoters are analysed, trying to identify the regulatory elements that are most important for the determination of the gene expression. Given the available data it wasn’t possible to draw definitive conclusions. In the end, few considerations on promoter engineering and complex circuits realization are presented. This section aims to briefly recall some of the problems outlined in the introduction and provide a few possible solutions.

De-authoring or Authoring?: A plunge into the question of authorship and language in J.M. Coetzee's Foe

J. M. Coetzee's Foe is not only a post-colonial novel, but it is also a re-writing of a classic, and its main themes are language, authorship, power and identity. Moreover, Foe is narrated by a woman, while written by a male, Nobel prize winning South African author. The aim of my tesina is to focus on the question of authorship and the role of language in Foe. Without any claim to be exhaustive, in the first section I will examine some selected extracts of Coetzee's book, in order to provide an analysis of the novel. These quotations will mainly be its metalinguistic parts and will be analysed in the “theory” sections of my work, relying on literary theory and on previous works on the novel. Among others, I will cover themes such as the relationship between speech and writing, the connection between writing, history, and memory, the role of silence and alternative ways of communicating and the relationship between literary authority and truth. These arguments will be the foundation for my second section, in which I will attempt to shed a light on the importance of the novel from a linguistic point of view, but always keeping an eye on the implication that this has on authorship. While it is true that it is less politically-permeated than Coetzee's previous works, Foe is above all a “journey of discovery” in the world of language and authorship. In fact, it becomes a warning for any person immersed in the ocean of language since, while everyone naturally tends to trust speech and writing as the only medium through which one can get closer to the truth, authority never is a synonym of reliability, and language is a system of communication behind which structures of power, misconceptions, lies, and treacherous tides easily hide.