Erschienen:
[Erscheinungsort nicht ermittelbar]: ETH Zurich, 2021
Sprache:
Englisch
Identifikator:
Entstehung:
Hochschulschrift:
Dissertation, ETH Zurich, 2021
Anmerkungen:
Beschreibung:
Synthetic biology applies an engineering mindset to the classical field of biology in order to facilitate the design of new proteins and cellular systems. Synthetic biology-inspired solutions and products are already a part of modern-day biotechnology and medicine. Drawing from genetic components from all kingdoms of life, engineers are able to design sophisticated genetic circuits that respond to various physical and chemical inputs One key motivation of synthetic biological engineering is the creation of tools to control cellular behavior typically through genetic circuits. However, the generation of gene circuits that respond to extracellular soluble ligands remains limited by the availability of suitable receptors. Although there is a vast number of endogenous receptors at hand, some proteins, especially disease markers, lack a native sensor module. To tackle this problem our group designed a novel receptor platform based on the established erythropoietin receptor scaffold that can accommodate various binding domains to detect small molecules as well as larger proteins. We took this idea further by employing a high-throughput selection for the generation of designed ankyrin repeat proteins (DARPins) to detect an even broader range of ligands in a streamlined fashion as described in chapter 1. We tested this pipeline by creating receptors that can detect sub-pathological concentrations of fibrin degradation products arising from e.g., thrombotic events. In order to detect a ligand without known receptor it is not necessarily the receptor that needs to be engineered – instead, the ligand itself can be modified to enable its detection. We show in chapter 2 how we designed an inducible gene switch that responds to the inert-to-human small molecule L-glucose. Here, we harnessed a metabolic process found in the soil bacterium Paraccocus species 43P to convert L-glucose into the active ligand of the bacterial transcriptional regulator LgnR. By fusing transcriptional modulator domains to LgnR, we could control gene expression from a synthetic promoter in an L-glucose responsive manner to enable production of the model biopharmaceutical rituximab. Physical stimuli like mechanical cues are omnipresent in our surroundings and aid cells to make informed decisions on propagation, tissue organization and cell fate. In chapter 3 we present results from our work on implementing a mechanically triggered gene switch in mammalian cells. With the help of a custom-made shear stress induction device we screened a small library of genetic circuits, transcription factors as well as accessory channels and receptors for their potential to improve shear stress induced calcium responses. This approach allowed us to identify key components that sensitize cells to mechanical cues to enable the exogenous control of gene expression. In chapter 4 we used the same device to study the effects of shear stress on lineage commitment of human induced pluripotent stem cells (hiPSCs) into the three germ layers. We were able to show that commitment to the ectoderm and mesoderm lineages was sensitive to shear stress through modulation of the Wnt/β-catenin signaling pathway while endoderm was not affected. More detailed analysis revealed that the effect is reversible and can be used to modulate gene expression levels and lineage commitment even in further matured cells of the mesoderm lineage.