Team:UCSF/Project/Circuit/Design

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<br><b>THIS PART IS FROM MODELING////WILL BE ALTERED<FONT COLOR="#008000">ASSUMPTIONS: </FONT COLOR="#008000"></b>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: <b><FONT COLOR="#008000"> 1) </font></b> protein degradation is linear;
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<br><b>THIS PART IS FROM MODELING////WILL BE ALTERED<FONT COLOR="#008000">ASSUMPTIONS: </FONT COLOR="#008000"></b>texttexttexttext.
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<b><FONT COLOR="#008000">2)  </font></b>protein production is based on a hill function and also depends on inducer concentration;
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<b><FONT COLOR="#008000">3)  </font></b> repression is governed by a hill function and depends on the concentration of dCas9 and gRNA complex; and
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<b><FONT COLOR="#008000">4)  </font></b> 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 <a href="http://en.wikipedia.org/wiki/Steady_State_theory#Quasi-steady_state" target="_blank">Quasi Steady State</a><span>).
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Revision as of 06:22, 27 September 2013

Decision Making Circuit

Building upon our CRISPR conjugation project, we began to think about what types of alterations we could confer to cells using the CRISPRi system. The unique capabilities of the CRISPR system allow for the design of a circuit that can achieve decision-making ability. 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.


Design Requirements: In order for this circuit to properly function, we had to address two main challenges:

1) identifying or constructing promoters that were differentially responsive to both high and low levels of an inducer

2) ensuring that the fluorescent proteins and gRNAs were produced in the same amount under the same promoter.

A large portion of our project was extensively testing and modeling promoter activity for use in the circuit, as well as designing a new lactose-reponsive promoter to sense changing levels of inducer.

To address the second issue, we made strategic design choices and utilized an RNA-cutting enzyme called Csy4 in order to equivalently express both the fluorescent protein and guideRNA for each part of the circuit. Both the protein and gRNA are behind the same promoter and linked together with a sequence coding a Csy4 cut site. After transcription, the RNA product is cleaved to make both mRNA for the fluorescent protein and the gRNA. To see more of our design strategies for the guideRNAs and using Csy4, please refer to our parts submitted to the registry.


THIS PART IS FROM MODELING////WILL BE ALTEREDASSUMPTIONS: texttexttexttext.

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