Detailed knowledge of the biological molecular mechanisms present inside the cell has revolutionized the idea of molecular biology by opening the doors to what has now become synthetic biology which considers an organism (e.g., bacteria, yeast) as a biological factory which can be adapted to a specific goal. Synthetic biology aims to apply key engineering concepts (e.g., automatic controls) to design and build new biological systems that exhibit predictable behavior. A fundamental tool of synthetic biology are specifically designed plasmid circuits which are a rapid way to rationally modify cellular metabolism according to a specific logic defined in the design phase. The function and, therefore, the construction of the circuit depends on two factors: the chosen microorganism and the purpose of the circuit. The limiting factor in new applications is finding a new microorganism and its characterization. It is a process that takes a lot of time and a lot of money. The solution to the latter problem would be to find a circuit that works on new organisms. This thesis was focused on both characterization of new CRISPR interference system obtained from S. aureus (SadCas9) and characterization of several parts of a new synthetic circuit aimed to maintain a constant expression of a gene of interest. Characterization of the circuit was done with computational approach. Particularly starting from a biological idea and a circuit construction, were obtained mathematical differential equations that were analyzed with several computational tools. For the characterization of the SadCas9 CRISPRi, first were assembled synthetic circuits that were used to get real biological data. This data was analyzed with MATLAB with a purpose of understanding the limits of the system.
La conoscenza sempre più dettagliata dei meccanismi molecolari biologici presenti all’interno della cellula ha rivoluzionato l’idea della biologia molecolare aprendo le porte a quella che oggi è diventata la biologia sintetica la quale considera un organismo (e.g., battere, lievito) come una fabbrica biologica che può essere adattata a un obiettivo specifico. La biologia sintetica mira ad applicare concetti chiave dell'ingegneria (e.g., controlli automatici) per progettare e costruire nuovi sistemi biologici che mostrano un comportamento predicibile. Uno strumento fondamentale della biologia sintetica sono i circuiti su plasmide che, progettati specificamente, sono un modo rapido per modificare razionalmente il metabolismo cellulare secondo una specifica logica definita in fase di design. Il funzionamento e, dunque, la costruzione del circuito dipende da due fattori: il microorganismo scelto e l’applicazione in cui si vuole inserire quest’ultimo. Il fattore limitante nelle nuove applicazioni è trovare un nuovo microorganismo e la sua caratterizzazione. È un processo che impiega tanto tempo e tanti soldi. La soluzione per quest’ultima problema sarebbe trovare un circuito che funzioni su nuovi organismi. Questa tesi si è concentrata sia sulla caratterizzazione del nuovo sistema di interferenza CRISPR ottenuto da S. aureus (SadCas9) sia sulla caratterizzazione di diverse parti di un nuovo circuito sintetico finalizzato a mantenere un'espressione costante di un gene di interesse. La caratterizzazione del circuito è stata eseguita con approccio computazionale. Partendo da un'idea biologica e da una costruzione circuitale, sono state ottenute equazioni differenziali matematiche che sono state analizzate con diversi strumenti computazionali. Per la caratterizzazione del SadCas9 CRISPRi, sono stati prima assemblati circuiti sintetici che sono stati utilizzati per ottenere dati biologici reali. Questi dati sono stati analizzati con MATLAB allo scopo di comprendere i limiti del sistema.
Computational biology approaches to design and characterize novel synthetic circuits based on the Staphylococcus aureus CRISPRi system
SHAPOSHNIKOV, ROMAN
2019/2020
Abstract
Detailed knowledge of the biological molecular mechanisms present inside the cell has revolutionized the idea of molecular biology by opening the doors to what has now become synthetic biology which considers an organism (e.g., bacteria, yeast) as a biological factory which can be adapted to a specific goal. Synthetic biology aims to apply key engineering concepts (e.g., automatic controls) to design and build new biological systems that exhibit predictable behavior. A fundamental tool of synthetic biology are specifically designed plasmid circuits which are a rapid way to rationally modify cellular metabolism according to a specific logic defined in the design phase. The function and, therefore, the construction of the circuit depends on two factors: the chosen microorganism and the purpose of the circuit. The limiting factor in new applications is finding a new microorganism and its characterization. It is a process that takes a lot of time and a lot of money. The solution to the latter problem would be to find a circuit that works on new organisms. This thesis was focused on both characterization of new CRISPR interference system obtained from S. aureus (SadCas9) and characterization of several parts of a new synthetic circuit aimed to maintain a constant expression of a gene of interest. Characterization of the circuit was done with computational approach. Particularly starting from a biological idea and a circuit construction, were obtained mathematical differential equations that were analyzed with several computational tools. For the characterization of the SadCas9 CRISPRi, first were assembled synthetic circuits that were used to get real biological data. This data was analyzed with MATLAB with a purpose of understanding the limits of the system.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12215