Revision as of 20:41, 1 November 2017

Design

Here you can read about the design principles that helped us structure, organize and execute our project. To read the story about how we developed the idea of CATE, go to Story of CATE. To skip to story and jump directly to how CATE is designed to treat tumors, see CATE in Action. For details about the circuit behind the story, visit our Circuit page.

Overview

On this page we explain the design principles we defined for our project and how we followed them. We structured our work in phases and tried to proceed through them. The phases apply to theoretical work (models) as well as to the practical (experiments). In first phase, we learned to handle the subjects and get familiar with the theory and literature. We designed, ordered and built constructs for tests of the experimental procedure and for further optimization. In the second phase, we tested predictions of the models and delivered parameters for new models with experiments. We optimized single parts to work in a regime where the model predicted the circuit to be functional.

We designed the project in a hierarchical bottom-up engineering approach: We divided the circuit into its different functions (F_a-F_e) and engineered them until they met our criteria.

Circuit Functions:

The individual constructs were assembled with molecular cloning and the functions were tested with reporter genes such as gfp and mcherry. Only if they behaved according to our requirements, we combined functions together. 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, thats why every function goes through the phases independently.

Plasmid creation during the CATE project

Phase I: Initial Design

In Phase I we decided for specific DNA sequences by reading up literature and planned the assembly of the parts into test devices. The test devices were then used to develop working assays.

Find the initial system designs of the Tumor Sensor and the Heat Sensor.

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.

Find out how we tested the quorum sensing to find the trigger point, on 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.
Read about the Heat Sensors RBS optimization process, 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 BFR 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.