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Title: Expanding molecular toolbox for synthetic biology
Author: Jajesniak, Pawel
ISNI:       0000 0004 7428 1554
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Synthetic biology can broadly be defined as a construction of new biological systems and modification of the existing ones. In recent years, synthetic biology has presented itself as a potential solution to many global challenges - e.g., climate change and scarcity of natural resources. The progress of synthetic biology is, however, largely reliant on the availability of a large repertoire of functional genetic parts. As a result, there exists an urgent need for enhancing and expediting the process of tapping into genetic diversity - both natural and laboratory-induced. Analysing the current landscape of synthetic biology and various approaches of accessing biological diversity, two high-impact areas, namely the basic molecular cloning and identification of genes encoding for complex microbial phenotypes, have been identified as particularly lacking and, as such, constitute the focal point of my PhD project. The culmination of the work presented in this dissertation are two molecular methods, QuickStep-Cloning and Multi-Genius, that aim to accelerate the development of synthetic biology. QuickStep-Cloning is a new molecular cloning technique that builds upon recent advances in megaprimer-based cloning to allow for seamless integration of a DNA fragment of interest into a plasmid in less than 6 hours - the result that could not be reproduced using state-of-the-art methods. The new improved version of the method, QuickStepS-Cloning, utilises phosphorothioate oligonucleotides to not only simplify the overall procedure but also significantly increase its cloning efficiency. It also shown that incorporating random mutagenesis into the method allows for streamlining directed evolution experiments. Whereas the potential applications of QuickStep-Cloning revolve around artificially-induced diversity, Multi-Genius builds upon the concept of genomic libraries to tap into naturally-existing diversity and expedite identification of genes encoding for useful phenotypes. The usefulness of the method has been proven by isolating thermotolerant variants of Escherichia coli DH5a and indentifying the gene responsible for the observed phenotype.
Supervisor: Wong, T. S. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available