Modeling and simulation of a synthetic genetic circuit that implements a multicelled behavior in a growing microcolony of E. coli

Synthetic Biology is a relatively new discipline, born at the beginning of the New Millennium, that brings the typical engineering approach (abstraction, modularity and standardization) to biotechnology. These principles aim to tame the extreme complexity of the various components and aid the construction of artificial biological systems with specific functions, usually by means of synthetic genetic circuits implemented in bacteria or simple eukaryotes like yeast. The cell becomes a programmable machine and its low-level programming language is made of strings of DNA. This work was performed in collaboration with researchers of the Department of Electrical Engineering of the University of Washington in Seattle and also with a student of the Corso di Laurea Magistrale in Ingegneria Biomedica at the University of Bologna: Marilisa Cortesi. During the collaboration I contributed to a Synthetic Biology project already started in the Klavins Laboratory. In particular, I modeled and subsequently simulated a synthetic genetic circuit that was ideated for the implementation of a multicelled behavior in a growing bacterial microcolony. In the first chapter the foundations of molecular biology are introduced: structure of the nucleic acids, transcription, translation and methods to regulate gene expression. An introduction to Synthetic Biology completes the section. In the second chapter is described the synthetic genetic circuit that was conceived to make spontaneously emerge, from an isogenic microcolony of bacteria, two different groups of cells, termed leaders and followers. The circuit exploits the intrinsic stochasticity of gene expression and intercellular communication via small molecules to break the symmetry in the phenotype of the microcolony. The four modules of the circuit (coin flipper, sender, receiver and follower) and their interactions are then illustrated. In the third chapter is derived the mathematical representation of the various components of the circuit and the several simplifying assumptions are made explicit. Transcription and translation are modeled as a single step and gene expression is function of the intracellular concentration of the various transcription factors that act on the different promoters of the circuit. A list of the various parameters and a justification for their value closes the chapter. In the fourth chapter are described the main characteristics of the gro simulation environment, developed by the Self Organizing Systems Laboratory of the University of Washington. Then, a sensitivity analysis performed to pinpoint the desirable characteristics of the various genetic components is detailed. The sensitivity analysis makes use of a cost function that is based on the fraction of cells in each one of the different possible states at the end of the simulation and the wanted outcome. Thanks to a particular kind of scatter plot, the parameters are ranked. Starting from an initial condition in which all the parameters assume their nominal value, the ranking suggest which parameter to tune in order to reach the goal. Obtaining a microcolony in which almost all the cells are in the follower state and only a few in the leader state seems to be the most difficult task. A small number of leader cells struggle to produce enough signal to turn the rest of the microcolony in the follower state. It is possible to obtain a microcolony in which the majority of cells are followers by increasing as much as possible the production of signal. Reaching the goal of a microcolony that is split in half between leaders and followers is comparatively easy. The best strategy seems to be increasing slightly the production of the enzyme. To end up with a majority of leaders, instead, it is advisable to increase the basal expression of the coin flipper module. At the end of the chapter, a possible future application of the leader election circuit, the spontaneous formation of spatial patterns in a microcolony, is modeled with the finite state machine formalism. The gro simulations provide insights into the genetic components that are needed to implement the behavior. In particular, since both the examples of pattern formation rely on a local version of Leader Election, a short-range communication system is essential. Moreover, new synthetic components that allow to reliably downregulate the growth rate in specific cells without side effects need to be developed. In the appendix are listed the gro code utilized to simulate the model of the circuit, a script in the Python programming language that was used to split the simulations on a Linux cluster and the Matlab code developed to analyze the data.

Interfaccia grafica per l'elaborazione di immagini microscopiche di colture batteriche geneticamente modificate

Le moderne tecniche di imaging e i recenti sviluppi nel campo della visione computazionale consentono sempre più diffusamente l'utilizzo di metodi di image analysis, specialmente in ambito medico e biologico, permettendo un maggiore supporto sia alla diagnosi, sia alla ricerca. Il lavoro svolto in questa tesi si pone in un contesto di ricerca di carattere interdisciplinare, e riguarda il progetto e la realizzazione di un‘interfaccia grafica per l'analisi di colture batteriche geneticamente modificate, marcate con proteine fluorescenti (GFP), acquisite tramite un microscopio ad epifluorescenza. Nota la funzione di risposta del sistema di acquisizione delle immagini, l'analisi quantitativa delle colture batteriche è effettuata mediante la misurazione di proprietà legate all'intensità della risposta al marcatore fluorescente. L'interfaccia consente un'analisi sia globale dei batteri individuati nell'immagine, sia di singoli gruppi di batteri selezionati dall'utente, fornendo utili informazioni statistiche, sia in forma grafica che numerica. Per la realizzazione dell'interfaccia sono state adottate tecniche di ingegneria del software, con particolare enfasi alla interazione uomo-macchina e seguendo criteri di usability, al fine di consentire un corretto utilizzo dello strumento anche da parte di personale senza conoscenza in campo informatico.

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.

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

Engineering the synthesis of lantibiotics in e. Coli by combining the cynnamicin and nisin modification systems.

I lantibiotici sono molecole peptidiche prodotte da un gran numero di batteri Gram-positivi, posseggono attività antibatterica contro un ampio spettro di germi, e rappresentano una potenziale soluzione alla crescente problematica dei patogeni multi-resistenti. La loro attività consiste nel legame alla membrana del bersaglio, che viene quindi destabilizzata mediante l’induzione di pori che determinano la morte del patogeno. Tipicamente i lantibiotici sono formati da un “leader-peptide” e da un “core-peptide”. Il primo è necessario per il riconoscimento della molecola da parte di enzimi che effettuano modifiche post-traduzionali del secondo - che sarà la regione con attività battericida una volta scissa dal “leader-peptide”. Le modifiche post-traduzionali anticipate determinano il contenuto di amminoacidi lantionina (Lan) e metil-lantionina (MeLan), caratterizzati dalla presenza di ponti-tioetere che conferiscono maggior resistenza contro le proteasi, e permettono di aggirare la principale limitazione all’uso dei peptidi in ambito terapeutico. La nisina è il lantibiotico più studiato e caratterizzato, prodotto dal batterio L. lactis che è stato utilizzato per oltre venti anni nell’industria alimentare. La nisina è un peptide lungo 34 amminoacidi, che contiene anelli di lantionina e metil-lantionina, introdotti dall’azione degli enzimi nisB e nisC, mentre il taglio del “leader-peptide” è svolto dall’enzima nisP. Questo elaborato affronta l’ingegnerizzazione della sintesi e della modifica di lantibiotici nel batterio E.coli. In particolare si affronta l’implementazione dell’espressione eterologa in E.coli del lantibiotico cinnamicina, prodotto in natura dal batterio Streptomyces cinnamoneus. Questo particolare lantibiotico, lungo diciannove amminoacidi dopo il taglio del leader, subisce modifiche da parte dell’enzima CinM, responsabile dell’introduzione degli aminoacidi Lan e MeLan, dell’enzima CinX responsabile dell’idrossilazione dell’acido aspartico (Asp), e infine dell’enzima cinorf7 deputato all’introduzione del ponte di lisinoalanina (Lal). Una volta confermata l’attività della cinnamicina e di conseguenza quella dell’enzima CinM, si è deciso di tentare la modifica della nisina da parte di CinM. A tal proposito è stato necessario progettare un gene sintetico che codifica nisina con un leader chimerico, formato cioè dalla fusione del leader della cinnamicina e del leader della nisina. Il prodotto finale, dopo il taglio del leader da parte di nisP, è una nisina completamente modificata. Questo risultato ne permette però la modifica utilizzando un solo enzima invece di due, riducendo il carico metabolico sul batterio che la produce, e inoltre apre la strada all’utilizzo di CinM per la modifica di altri lantibiotici seguendo lo stesso approccio, nonché all’introduzione del ponte di lisinoalanina, in quanto l’enzima cinorf7 necessita della presenza di CinM per svolgere la sua funzione.