Team:ETH Zurich/Design
Design
Here you can read about the design principles that helped us structure, organize and execute our project. To read about the story of how we developed the idea of CATE, go to Story of CATE. To skip this story and jump directly to how CATE is designed to treat tumors, see CATE in Action. For details about the circuit behind the functioning, visit our Circuit page.
Overview
We structured our work in phases and gradually proceeded through them. The phases apply to theoretical (models) as well as practical (experiments) work. In phase one, we get familiar with the details of the respective subjects. Based on existing data, we designed, ordered and built constructs for experimental procedures and further optimization. In phase two, we tested predictions of the models and generated data to fit their parameters. Optimization of single parts was guided by theoretical work in order to achieve functioning parts.
We designed the project in a hierarchical bottom-up engineering approach: We divided the circuit into its different functions (Fa-Fe) and engineered them until they met our criteria.
Circuit Functions:
The individual constructs were assembled by various molecular cloning techniques. Subsequently, functions were assessed with reporter genes such as sfGFP and mCherry. Only if they behaved according to our requirements, we coupled different functions. In parallel, we ordered the full genetic circuit of CATE with restriction sites along the critical loci in order to rapidly exchange promotors, ribosome binding sites or coding sequences after we experimentally optimized the parts.
We worked in parallel on the functions of CATE, which is why every function goes through the phases independently.
Phase I: Initial Design
In Phase I we considered previous work in order to design specific DNA sequences. Subsequently, we planned assembly of the parts into test devices. These were then used to develop assays that can be used to characterize the parts in vitro.
Phase II: Tests and Optimization
In this phase the assays have been developed and show us if the function behaves as expected. At this point, we could therefore start to tune the functions. This was achieved by changing the expression levels of proteins with RBS libraries or different designs of a promotor.
