Expanding the regulatory repertoire available for synthetic genetic circuits in S. cerevisiae

• Main Author: Weenink, Tim
• Other Authors: Ellis, Tom
• Published: Imperial College London 2016
• Subjects: 572.8
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.726929

Complexity is arguably the biggest challenge to the field of synthetic biology today. As synthetic constructs include more and more parts, their performance becomes less predictable and more costly to host cells. In this thesis, we work towards the expansion of the regulatory repertoire available for S. cerevisiae with the aim of reducing the complexity of synthetic gene circuits and thus improving their performance. Three projects contribute to achieving this goal. First, we note that no tool exists for yeast similar to the bacterial RBS Calculator that enables accurate tuning of expression levels. We address this by designing a system for tuning translation efficiency based on predictable hairpin structures placed in mRNA 5’UTRs. We characterise the relationship between folding strength and expression output and show that this facilitates predictable expression level tuning. We implement this system as a method for rapid library generation and characterise it with regards to both context and predictability. Next, we implement transcriptional interference (TI) as a tool to augment existing regulatory interactions that may not possess sufficient regulatory power to implement the desired function. We demonstrate that TI performs as expected at the mRNA level, but observe that the imple- mentation interferes with translational output. We test a variety of solutions relying on different molecular mechanisms within the host and conclude with a system for functionalising the RNA product produced. In the third project, we implement a system for simplifying circuit designs by combining activation and repression functionality into a single transcription factor. This system is based on TAL-effectors fused to an activation domain that can be targeted to an upstream region of a promoter for activation and a downstream region for repression. In a systematic series of characterisation experiments we show the creation of a TAL-effector promoter pair that exhibits the desired functionality.