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Embargoed until 2025-06-01
Author
Date
2023Type
- Doctoral Thesis
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Abstract
S. cerevisiae is an organism that has, behind the scenes, shaped our modern civilization. Beyond its culinary applications, it has contributed greatly to our understanding of eukaryotic organisms at the molecular level. Given its tractability, it has been an instrumental model organism in investigating gene function and gene-gene interactions. As it shares a large number of homologous genes with other eukaryotic cells, the knowledge gained in yeast has proven invaluable when trying to understand gene function and gene networks in higher organisms. Additionally, yeast is routinely used in laboratory evolution, whereby proteins acquire novel functions useful for basic research, and industrial and pharmaceutical applications. Yet to this day there is still a lot that is unknown about yeast genes and proteins, and we are still some ways off from harvesting the full potential of yeast as an evolution engine. In this thesis, we develop genetic tools to exploit yeast both as a genetic discovery tool and as a novel protein function evolver. A gene’s function is investigated through perturbations of its protein product. In theory, inducible systems provide the largest range of possible perturbations, from complete absence to overexpression. Yet, even in our well-studied model organism there are no inducible systems that could cover this range while also allowing precise titration at intermediate expression levels under different growth conditions.
In Chapter 2, we create one such inducible system: the Well-tempered Controller 846 (WTC846). It is based on a highly active, endogenous yeast promoter that we engineered to be fully repressed by the prokaryotic DNA binding protein TetR. Gene expression is then induced by the small molecule anhydrotetracycline. WTC846 also contains a negative feedback loop, whereby TetR represses its own expression. We demonstrate that these components allow WTC846 to bring about reversible knockouts, overexpression and very precise titration of any protein in yeast in any growth condition. Yet genes and their protein products do not function in isolation. They interact with other proteins to form pathways and networks, from which emerge the complex phenotypes we see in cells, such as the precisely regulated cell cycle. Therefore, understanding gene-gene interactions is paramount to understanding how the cell functions. As with investigations of a single gene, perturbations can inform us about gene-gene interactions. But for this, orthogonal systems that can perturb interacting genes separately are required.
In Chapter 3, we extend the WTC846 system to two new repressor-inducer pairs to provide a total of three such orthogonal systems. WTC847 is based on the repressor LexA-human estrogen receptor fusion and is induced by β-estradiol. WTC848 is based on the prokaryotic repressor LacI and is induced by IPTG. While the two new WTC systems do not have the range afforded to the experimenter by WTC846, we demonstrate that they can nevertheless be useful for revealing gene-gene interactions in yeast. We use the three systems to simultaneously and orthogonally perturb genes within the anaphase signaling pathway and reveal dosage-dependent interactions. One complex phenotype that all living organisms display is their ability to evolve and adapt. This happens through mutations and natural selection at incredibly long timescales, during which beneficial mutations give cells a growth advantage over others. We can artificially reduce this time in the laboratory by increasing mutations rates, but if the phenotype created by the mutants cannot be linked to a growth advantage, screening limits the range of solutions accessible to the experimenter. Especially in the case of binding interactions, for example between a receptor and a ligand, growth-based selection systems do not exist.
In Chapter 4, we co-opt components of the WTC systems to create a closed-loop, in vivo evolution system in yeast, which reliably links strength of binding interactions to a growth advantage. We use this genetic circuit, called the Evolverator, to evolve both a receptorhormone ligand interaction, and a DNA binding protein-DNA interaction, reveling thus far uncharacterized mutations. Show more
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https://doi.org/10.3929/ethz-b-000614657Publication status
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Publisher
ETH ZurichSubject
Synthetic Biology; Yeast; Transcriptional Control; Closed-loop evolution; Mutagenesis; Genetic DiscoveryOrganisational unit
03699 - Stelling, Jörg / Stelling, Jörg
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