Synthetic Biology is a branch of Bioengineering that combines biological, statistical and engineering principles. Established in the early 2000s, its main application lies in implementing pathways in organisms of interest. To ensure their proliferation under controlled laboratory conditions, a suitable microbiological culture method is of paramount importance. The types of culture currently employed differ according to the way in which the medium, containing the nutrients for cell growth, is delivered. Continuous culture, the technique featured in this study, allows one to work with viable, metabolically active organisms over extended periods of time. Among the tools used to implement this strategy are bioreactors. They work to create a favourable environment for microbial growth. There are platforms that can control the activity of several bioreactors simultaneously, allowing different experimental procedures to be conducted in parallel. One of these is called Chi.Bio, developed at Oxford University. Its affordability and sensor system make it potentially useful for small- to medium-scale experiments. Chi.Bio allows real-time monitoring of the state of the culture through measurements of optical density, fluorescence and temperature. For a correct set-up of the experimental conditions to which the organisms were to be subjected, the first part of the study focused on the correct calibration of the instrument. Therefore, it was necessary to select recombinant strains that constitutively or inducibly expressed proteins used as fluorescent reporters. The second part of the project focused on the analysis of recombinant strains containing different synthetic circuits. Different constructs were tested in order to characterise their response to different cellular stress conditions, and to ascertain that the results were in line with those obtained with other experimental methodologies (population measurements in batch and single-cell experiments in microfluidic devices). The experiments were conducted not only by using the platform as a turbidostat, but also by implementing customised programmes in Python language to control other parameters besides optical density. The thesis work was carried out at the Bioinformatics and Synthetic Biology laboratory and the laboratory activities at the CHT Cell Culture Laboratory, Centre for Health Technologies.
La Biologia Sintetica è una branca della Bioingegneria che coniuga principi biologici, statistici e ingegneristici. Nata nei primi anni duemila, la sua principale applicazione consiste nell'implementare pathway di interesse in organismi di interesse. Per garantirne la proliferazione in condizioni di laboratorio controllate, è di fondamentale importanza adottare un metodo di coltura microbiologica adatto. I tipi di coltura attualmente impiegati differiscono in base alle modalità con cui il terreno, contenente i nutrienti per la crescita cellulare, viene erogato. La coltura continua, tecnica protagonista di questo studio, permette di lavorare, per tempi prolungati, con organismi vitali e metabolicamente attivi. Tra gli strumenti impiegati per attuare tale strategia vi sono i bioreattori. Essi lavorano per creare un ambiente favorevole alla crescita microbica. Esistono piattaforme in grado di controllare l'attività di più bioreattori contemporaneamente, permettendo condurre differenti procedure sperimentali parallelamente. Una di queste prende il nome di Chi.Bio, sviluppata alla Oxford University. L'accessibilità in termini di costo e il sistema di sensori di cui è dotata la rendono potenzialmente utile per esperimenti di piccola-media scala. Chi.Bio permette di monitorare in tempo reale lo stato della coltura tramite misure di densità ottica, fluorescenza e temperatura. Per un corretto set-up delle condizioni sperimentali a cui sottoporre gli organismi, la prima parte dello studio si è focalizzata sulla corretta calibrazione dello strumento. Pertanto è stato necessario selezionare dei ceppi ricombinanti che esprimessero in maniera costitutiva o inducibile proteine usate come reporter fluorescenti. La seconda parte del progetto si è focalizzata sull'analisi di ceppi ricombinanti contenenti diverse circuiterie sintetiche. Sono stati testati diversi costrutti col fine di caratterizzare la loro risposta a diverse condizioni di stress cellulare, e di constatare che i risultati fossero conformi a quanto ottenuto con altre metodologie sperimentali (e.g. misure di popolazione in esperimenti batch e singola-cellula in dispositivi microfluidici). Gli esperimenti sono stati condotti non solo utilizzando la piattaforma come un turbidostato, ma anche implementando, in linguaggio Python, programmi personalizzati che controllassero altri parametri oltre alla densità ottica. Il lavoro di tesi è stato svolto presso il laboratorio di Bioinformatica e Biologia Sintetica e le attività di laboratorio presso il laboratorio di colture cellulari CHT, Centre for Health Technologies.
Valutazione tecnica di una piattaforma scalabile di bioreattori per lo studio di colture batteriche continue.
MORDEGLIA, GIULIA
2023/2024
Abstract
Synthetic Biology is a branch of Bioengineering that combines biological, statistical and engineering principles. Established in the early 2000s, its main application lies in implementing pathways in organisms of interest. To ensure their proliferation under controlled laboratory conditions, a suitable microbiological culture method is of paramount importance. The types of culture currently employed differ according to the way in which the medium, containing the nutrients for cell growth, is delivered. Continuous culture, the technique featured in this study, allows one to work with viable, metabolically active organisms over extended periods of time. Among the tools used to implement this strategy are bioreactors. They work to create a favourable environment for microbial growth. There are platforms that can control the activity of several bioreactors simultaneously, allowing different experimental procedures to be conducted in parallel. One of these is called Chi.Bio, developed at Oxford University. Its affordability and sensor system make it potentially useful for small- to medium-scale experiments. Chi.Bio allows real-time monitoring of the state of the culture through measurements of optical density, fluorescence and temperature. For a correct set-up of the experimental conditions to which the organisms were to be subjected, the first part of the study focused on the correct calibration of the instrument. Therefore, it was necessary to select recombinant strains that constitutively or inducibly expressed proteins used as fluorescent reporters. The second part of the project focused on the analysis of recombinant strains containing different synthetic circuits. Different constructs were tested in order to characterise their response to different cellular stress conditions, and to ascertain that the results were in line with those obtained with other experimental methodologies (population measurements in batch and single-cell experiments in microfluidic devices). The experiments were conducted not only by using the platform as a turbidostat, but also by implementing customised programmes in Python language to control other parameters besides optical density. The thesis work was carried out at the Bioinformatics and Synthetic Biology laboratory and the laboratory activities at the CHT Cell Culture Laboratory, Centre for Health Technologies.| File | Dimensione | Formato | |
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Descrizione: Valutazione tecnica di una piattaforma scalabile di bioreattori per lo studio di colture batteriche continue.
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https://hdl.handle.net/20.500.14239/33345