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Team:UCSF/Project/Circuit/Design
From 2013.igem.org
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| - | <br><FONT COLOR="#008000"><u>Design Requirements: </FONT COLOR="#008000"></u><br>In order for this circuit to properly function, we had to address two main challenges: <br><br><center><b><FONT COLOR="#008000"> 1) </font></b> identifying or constructing promoters that were differentially responsive to both <b>high</b> and <b>low</b> levels of an inducer</center> | + | <br><FONT COLOR="#008000"><u>Design Requirements: </FONT COLOR="#008000"></u><br> |
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| + | In order for this circuit to properly function, we had to address two main challenges: <br><br><center><b><FONT COLOR="#008000"> 1) </font></b> identifying or constructing promoters that were differentially responsive to both <b>high</b> and <b>low</b> levels of an inducer</center> | ||
<br><center><b><FONT COLOR="#008000">2) </font></b> ensuring that the fluorescent proteins and gRNAs were produced in the same amount under the same promoter.</center> | <br><center><b><FONT COLOR="#008000">2) </font></b> ensuring that the fluorescent proteins and gRNAs were produced in the same amount under the same promoter.</center> | ||
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<br><br>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 <a href="https://2013.igem.org/Team:UCSF/Parts">registry</a>. | <br><br>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 <a href="https://2013.igem.org/Team:UCSF/Parts">registry</a>. | ||
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| - | <br><FONT COLOR="#008000"><u>Using CRISPR to Create Scalable Circuits: </u></FONT COLOR="#008000"></b> | + | <br><FONT COLOR="#008000"><u>Using CRISPR to Create Scalable Circuits: </u></FONT COLOR="#008000"></b> |
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| + | A novel feature of our synthetic circuit are the infinite designs that stem from using gRNA’s rather than repressors. These gRNA’s allow for high specificity to DNA sequences, are easily manufactured, and allow for numerous decision making circuits. We can create multiple plasmids featuring these gRNA’s and insert these plasmids, as well as a plasmid with dCAS9, into an organism. From there these gRNA’s will combine with dCAS9 only when the appropriate chemical signal starts transcription of these gRNA’s. In addition to Plasmid A, we have started constructing Plasmid B which will feature pigments rather than fluorescent proteins. | ||
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Revision as of 22:18, 27 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. 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:
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.
Using CRISPR to Create Scalable Circuits:
A novel feature of our synthetic circuit are the infinite designs that stem from using gRNA’s rather than repressors. These gRNA’s allow for high specificity to DNA sequences, are easily manufactured, and allow for numerous decision making circuits. We can create multiple plasmids featuring these gRNA’s and insert these plasmids, as well as a plasmid with dCAS9, into an organism. From there these gRNA’s will combine with dCAS9 only when the appropriate chemical signal starts transcription of these gRNA’s. In addition to Plasmid A, we have started constructing Plasmid B which will feature pigments rather than fluorescent proteins.
