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 characterize the parts in vitro.
Phase II: Tests and Optimization
In this phase the assays work and show us if the function behaves as expected. We could therefore start to tune the functions by changing the expression level of proteins with RBS libraries or different designs of a promotor. Because of time restrictions we did not go into protein engineering.
- We tested the quorum sensing to find the trigger point, at which it activates the AND-gate promoter, or the dose-response of different AND-gate promoter designs.
- The initial model was fitted with the experimental data and helped us design the next experiment. Read more about how the model was fitted here.
- We optimized the Heat Sensor's RBS to reduce the leakiness of the promoter and make it possible to control protein E (which is very toxic for cells, and would kill them immediately if regulated by a leaky promoter).
- We measured the AHL dose-response of the bacterioferriting regulating promoter to make sure the promoter is actively inducible.
- In the same way, we characterized the azurin producing test device.
- We created a protein E RBS library to find variants able to be regulated by the heat sensor (without immediate killing of the cell).
- We modeled the heat diffusion of 45 °C for 3 h in a tumor to find out if it is acceptable for the tumor surrounding tissue, because the heat sensors detection temperature was 45 °C, not 42 °C as initially planned.
Phase III: Demonstration of the function
Important experiments that show our system at work were performed with biological triplicates. The assays were kept the same as in phase II and Protocols are available. Find the important results summarized on the Results page.
