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.

Controllo ottico automatizzato di circuiti fotonici integrati: Progettazione, realizzazione e valutazione

In this report a new automated optical test for next generation of photonic integrated circuits (PICs) is provided by the test-bed design and assessment. After a briefly analysis of critical problems of actual optical tests, the main test features are defined: automation and flexibility, relaxed alignment procedure, speed up of entire test and data reliability. After studying varied solutions, the test-bed components are defined to be lens array, photo-detector array, and software controller. Each device is studied and calibrated, the spatial resolution, and reliability against interference at the photo-detector array are studied. The software is programmed in order to manage both PIC input, and photo-detector array output as well as data analysis. The test is validated by analysing state-of-art 16 ports PIC: the waveguide location, current versus power, and time-spatial power distribution are measured as well as the optical continuity of an entire path of PIC. Complexity, alignment tolerance, time of measurement are also discussed.

Virtual Calibration of CO2 and Pollutant Emissions of a High-Performance PHEV using Model-in-the-Loop Methodology

The study analyses the calibration process of a newly developed high-performance plug-in hybrid electric passenger car powertrain. The complexity of modern powertrains and the more and more restrictive regulations regarding pollutant emissions are the primary challenges for the calibration of a vehicle’s powertrain. In addition, the managers of OEM need to know as earlier as possible if the vehicle under development will meet the target technical features (emission included). This leads to the necessity for advanced calibration methodologies, in order to keep the development of the powertrain robust, time and cost effective. The suggested solution is the virtual calibration, that allows the tuning of control functions of a powertrain before having it built. The aim of this study is to calibrate virtually the hybrid control unit functions in order to optimize the pollutant emissions and the fuel consumption. Starting from the model of the conventional vehicle, the powertrain is then hybridized and integrated with emissions and aftertreatments models. After its validation, the hybrid control unit strategies are optimized using the Model-in-the-Loop testing methodology. The calibration activities will proceed thanks to the implementation of a Hardware-in-the-Loop environment, that will allow to test and calibrate the Engine and Transmission control units effectively, besides in a time and cost saving manner.

Novel Machine-Learning Based IC Testing Strategy

The final goal of the thesis should be a real-world application in the production test data environment. This includes the pre-processing of the data, building models and visualizing the results. To do this, different machine learning models, outlier prediction oriented, should be investigated using a real dataset. Finally, the different outlier prediction algorithms should be compared, and their performance discussed.