Towards Iterative Simultaneous Yeast Display: a novel peptide biosensor platform

Biotechnology and medical industries currently lack the necessary range of accurate sensors for rapidly detecting low concentration analytes. Biosensors can be used to mitigate the limitations of current sensing technologies by facilitating accurate, high-throughput detection assays. However, rational biosensor engineering is limited by the range of known protein binding domains that can be used to detect molecules of interest. Furthermore, peptide selection techniques such as phage- and yeast-display are limited in their suitability for biosensor development, as the resulting single binding peptides are difficult to integrate into generic biosensor architecture, and target molecules must be modified for the assay. Using a novel combination of yeast-display and survival-based screening, a process termed Iterative Simultaneous Yeast Display (ISYD) is proposed to generate binding peptides for biosensors. This thesis verified and optimised various necessary concepts for ISYD to function including; the use of introns as a modular, scarless peptide assembly method, and the optimisation of molecular biology techniques to maximise peptide library structure and diversity. This thesis enabled some of the core concepts of ISYD to be established, with a parallel focus on laboratory automation of molecular biology that will improve the repeatability and throughput of the ISYD technique.