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Team:UCSF/Project/Circuit/Design1
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<span>CRISPRi Conjugation</span> | <span>CRISPRi Conjugation</span> | ||
| - | <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Design1">Design</a> | + | <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Design1">Project Design</a> |
<a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Data1">Data</a> | <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Data1">Data</a> | ||
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<span>CRISPRi Circuit</span> | <span>CRISPRi Circuit</span> | ||
| - | <a href="/Team:UCSF/Project/Circuit/ | + | <a href="/Team:UCSF/Project/Circuit/Design1">Circuit Design</a> |
| - | <a href="/Team:UCSF/Project/Circuit/ | + | <a https://2013.igem.org/Team:UCSF/Project/Conjugation/Promoter1">Promoter Engineering</a> |
| + | <a href="/Team:UCSF/Project/Circuit/Data1">Data</a> | ||
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<p2>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 <FONT COLOR="#008000"><b>we utilize guideRNAs (gRNAs) in lieu of repressors, which will allow for a highly scalable design. </b></FONT COLOR="#008000"><br><br></p2> | <p2>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 <FONT COLOR="#008000"><b>we utilize guideRNAs (gRNAs) in lieu of repressors, which will allow for a highly scalable design. </b></FONT COLOR="#008000"><br><br></p2> | ||
<p2>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 whether a high or low amount of chemical signal (inducer) is present, the cells would produce either RFP or GFP. The graph below is a model we created that shows our desired output based on inducer concentration.</p2> | <p2>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 whether a high or low amount of chemical signal (inducer) is present, the cells would produce either RFP or GFP. The graph below is a model we created that shows our desired output based on inducer concentration.</p2> | ||
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<p2><br>So why would we want a circuit to be transferred to a specific organism? Well our digestive trait is home to almost 1000 different species that have shown to directly affect our health and well-being. To improve and maintain healthy living it would be useful to have the ability to change the microbial community. For example, if a large of amount of a certain sugar was present in your gut ("signal #1") you might want to slow the growth of a certain bacteria to prevent a harmful outcome. In another scenario ("signal #2") it might be useful to increase the growth of other specific bacteria in your gut. </p2> | <p2><br>So why would we want a circuit to be transferred to a specific organism? Well our digestive trait is home to almost 1000 different species that have shown to directly affect our health and well-being. To improve and maintain healthy living it would be useful to have the ability to change the microbial community. For example, if a large of amount of a certain sugar was present in your gut ("signal #1") you might want to slow the growth of a certain bacteria to prevent a harmful outcome. In another scenario ("signal #2") it might be useful to increase the growth of other specific bacteria in your gut. </p2> | ||
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<h3>Design Requirements:</h3> | <h3>Design Requirements:</h3> | ||
<p2>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> | <p2>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. | + | <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>A large portion of our project was extensively <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">testing</a> and <a href="https://2013.igem.org/Team:UCSF/Modeling">modeling</a> promoter activity for use in the circuit, as well as designing a new <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">lactose-reponsive</a> promoter to sense changing levels of inducer. We created these promoters by altering the location and number of repressor binding sites in the promoter region. | <br>A large portion of our project was extensively <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">testing</a> and <a href="https://2013.igem.org/Team:UCSF/Modeling">modeling</a> promoter activity for use in the circuit, as well as designing a new <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">lactose-reponsive</a> promoter to sense changing levels of inducer. We created these promoters by altering the location and number of repressor binding sites in the promoter region. | ||
<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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Latest revision as of 03:55, 29 October 2013
CRISPR Decision-Making Circuit
Promoter Sensitivity:
So why would we want a circuit to be transferred to a specific organism? Well our digestive trait is home to almost 1000 different species that have shown to directly affect our health and well-being. To improve and maintain healthy living it would be useful to have the ability to change the microbial community. For example, if a large of amount of a certain sugar was present in your gut ("signal #1") you might want to slow the growth of a certain bacteria to prevent a harmful outcome. In another scenario ("signal #2") it might be useful to increase the growth of other specific bacteria in your gut.
CRISPRi Circuit Design:
In scenario 1, 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-gRNA complex will then repress the RFP (left panel). In scenario 2 the inverse follows the same principle. If suddenly the chemical signal increases to a high amount, RFP and gRNA to GFP will be produced. The gRNA-dCas9 complex will then repress GFP expression (right panel).
Design Requirements:
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. We created these promoters by altering the location and number of repressor binding sites in the promoter region.
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:
