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Team:UCSF/Project/Circuit/Design
From 2013.igem.org
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Revision as of 22:44, 20 September 2013
Building upon our CRISPR conjugation project, we began to think about what types of alterations we could confer to cells using the CRISPRi system. One alteration we had in mind was a decision making ability that can be achieved with a circuit design. Many synthetic circuits have been created using multiple repressors as their switch. In our circuit design we utilize guideRNAs (gRNAs) in lieu of repressors which will allow for a highly scalable design.
Our synthetic circuit has been engineered to give cells a decision making ability between differential outputs and will utilize CRISPRi as a switching mechanism between these outputs. Depending on a high or low amount of chemical signal, our cells can produce either RFP or GFP. If there was a low amount of chemical signal, GFP and a gRNA to RFP will be produced. The gRNA will then combine with dCas9, which has been incorporated on a separate plasmid. The dCas9 will then repress the RFP. The inverse follows the same principle; if suddenly the chemical signal increases, RFP and gRNA to GFP will be produced. The gRNA will combine with dCas9 and repress GFP expression.
If we can get this result from our model, then it would help us figure out how much inducer to add to our experiments in order to get the desired result.
Before getting into any modeling, we had to first figure out what the design of the synthetic circuit would be. It’s essentially the same diagram as the one shown on the synthetic circuit page (link here), but we added letters to represent each variable for our model.
(R:C – gRNA/dCas9 complex; R – gRNA; C – dCas9; L – low inducible promoter; H – high inducible promoter)
(R:C – gRNA/dCas9 complex; R – gRNA; C – dCas9; L – low inducible promoter; H – high inducible promoter)
ASSUMPTIONS: While creating the model for our system, we made a few assumptions about some of the aspects of the model that would be impossible for us to know within a few months. We made four assumptions: 1) protein degradation is linear; 2) protein production is based on a hill function and also depends on inducer concentration; 3) repression is governed by a hill function and depends on the concentration of dCas9 and gRNA complex; and 4) that the binding and unbinding of dCas9 and gRNA complex happens much faster than the production/degradation of gRNA and fluorescent proteins (the complex is at Quasi Steady State).
