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Title: Development of phage-assisted evolution and riboregulation strategies
Author: Serrano, Eduardo Goicoechea
ISNI:       0000 0004 9358 2846
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2019
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Riboswitches are short RNA sequences that undergo a structural modification in the presence of a specific molecule, regulating gene expression in the process. A few examples of riboswitches have been described in eukaryotes, but they are present in many species of bacteria. These molecules are of great interest in synthetic biology for their inductive regulatory properties, which could be used in metabolic studies, or as biosensors in medical, industrial or environmental contexts. However, riboswitches are very substrate-specific, creating a problem for their use with novel compounds; and current development procedures suffer from issues such as being too laborious and not using in vivo conditions. By developing strategies based on Phage Assisted Continuous Evolution (PACE) and using T7 phage along with a double selection system, we aim to develop a way of obtaining riboswitches that show improvements compared to a control sequence or with a different specificity. This procedure could even be used to develop novel riboswitches not present in nature. Once the method is properly established, the strategy could be used not only for the directed evolution of riboswitches, but also other types of sensors, such as protein or RNA receptors. Alternative evolution strategies based on phage transduction were also tested, along with inducible RNA systems based on the phage Qb. These three processes have yielded different results, namely a randomised library of theophylline riboswitch sequences in phages, a working transduction system, and an inducible RNA plasmid, respectively. In the first case, by using a double selection process to achieve evolution, we have shown sequence variation within the phage populations progressively decreased over time, until a single sequence was prominently represented in the population. The sequence showed higher activation folds at a lower concentration of the activating molecule than in initial generations, taken as an indication that evolution had taken place. This library was also adapted into the transduction strategy thanks to the use of a working T7 phage packaging signal. As for the RNA-based plasmid system, we have shown it to be an inducible, transient expression system that could be used as a novel way to regulate gene expression and bypass CRISPR systems. This set of results speak of the possibilities held by these strategies, not just for their specific areas of research, but for synthetic biology at large, from bioremediation to treatment.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology ; QP Physiology ; QR Microbiology