De novo designed protein-protein interaction domains for synthetic biology applications in cells

  • Caitlin L Edgell

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Synthetic biology aims to make the design of novel biological phenomena easier, faster and more precise through the design and engineering of biological components. This can be attempted using “top-down” approaches through engineering the genomes of extant organisms. Alternatively, it can be performed in a “bottom-up” fashion, by designing the characteristics of individual molecules and combining them in increasingly complex systems. The latter allows for the design of entirely new behaviours through the assembly of components that are not observed in nature. Important design targets for this bottom-up approach are those that control the expression of genes, that is, transcription factors.
Transcription factors are ubiquitous and highly diverse. They are also the first point of control for the production of all other cellular components, both natural and designed. In prokaryotes, transcriptional control can be achieved by actively recruiting RNA polymerase to a gene of interest via protein-protein interactions or preventing RNA polymerase from binding to DNA by assembling oligomers at a promoter. Therefore, in their simplest forms, both transcriptional activators and repressors require the ability to (1) bind to DNA and (2) bind to other proteins. By combining protein domains that perform these functions entirely artificial transcription factors can be generated. In the past, this has been achieved by combining naturally occurring protein domains. However, advances in rational protein design mean we can now design these domains de novo.
Herein, a set of tetrameric coiled coil-based protein-protein interaction domains is rationally designed and characterised in vitro. These coiled coils are then used to direct protein-protein interactions in E. coli, demonstrated through the design of transcriptional regulators which can themselves be controlled at the transcriptional level. In the future, these constitutively interacting domains could be made even more useful through the incorporation of dynamic behaviours, such as ligand-induced conformational changes.
Date of Award23 Jan 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorDek N Woolfson (Supervisor) & Nigel J Savery (Supervisor)

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