Difference between revisions of "Team:ETH Zurich/Design"
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| − | <p>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. | + | <p>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 <a href="https://2017.igem.org/Team:ETH_Zurich/Experiments/Tumor_Sensor#phaseI">Tumor Sensor</a> and the <a href="https://2017.igem.org/Team:ETH_Zurich/Experiments/Heat_Sensor#phaseI">Heat Sensor</a>. |
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Revision as of 00:16, 1 November 2017
Design
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 (Fa-Fe) 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.
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. We created reduced libraries (Red Libs) to create variants with an expression level that fits the needs of the circuit. As soon as the parts met our requirements, we proceeded to combine them to more complex circuits in phase III.
Phase III
If two functions were ready to be combined, phase III experiments were initiated. The assays were kept the same as in phase II.
Phase IV - proof of concept
This is the final phase and important features of CATE are shown to work in phase IV experiments.
Experiments Plan
Here you find the overview of the phases of our project and which parts were used at which time.
