Team:UCSF/Project/Circuit/Design1

From 2013.igem.org

(Difference between revisions)
Line 508: Line 508:
<div id="leftcontenttext" style = "width: 740px;height:320px" align="justify">
<div id="leftcontenttext" style = "width: 740px;height:320px" align="justify">
-
<h3>What is conjugation? </h3>
+
<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></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>
-
<h3>Combining CRISPRi and Conjugation</h3>
+
-
<p2>By combining CRISPRi and conjugation, we've come up with a system that will allow us to specifically target certain populations within a microbiome. To do this, an engineered cell capable of conjugating must be introduced into a microbiome of interest. The engineered cell, or donor cell, is capable of conjugating (proteins necessary for conjugation are contained in the genome) and carries a conjugative plasmid, which codes for a catalytically dead Cas9 (dCas9) protein and guide RNA (gRNA) for a specific gene that is present in the targeted population.</p2>
+
</div>
</div>
<div id="photos" style = "width: 740px" align="justify">
<div id="photos" style = "width: 740px" align="justify">
<center><img style="margin-top:0px; height:280px"; padding:0;"
<center><img style="margin-top:0px; height:280px"; padding:0;"
-
src="https://static.igem.org/mediawiki/2013/a/ac/UCSF2013-Project-Conjugation-1.png"> </center>
+
src="https://static.igem.org/mediawiki/2013/3/3f/DesiredOutput-UCSF.jpg"> </center>
</div>
</div>
<div id="leftcontenttext" style = "width: 740px; height:95px" align="justify">
<div id="leftcontenttext" style = "width: 740px; height:95px" align="justify">
-
<p2><br>Upon conjugation with the target population, the conjugative plasmid would be transferred. Both dCas9 and gRNA would subsequently be expressed in the recipient cell, and the complex formed will repress the targeted gene specified by the gRNA, shutting down certain cell functions. </p2>
+
<h3>Promoter Sensitivity:</h3>
 +
<p2>To specify our outputs we want a promoter that responds to a low amount of inducer and a promoter that responds to a high amount of inducer. </p2>
</div>
</div>
-
<div id="photos" style = "width: 740px" align="justify">
+
<div id="photos" style = "width: 300px; float:left" align="justify">
<center><img style="margin-top:0px; height:250px"; padding:0;"
<center><img style="margin-top:0px; height:250px"; padding:0;"
-
src="https://static.igem.org/mediawiki/2013/8/83/Conjugation-2.png"> </center>
+
src="https://static.igem.org/mediawiki/2013/4/40/UCSF_LowPromoterModel.jpg"> </center>
</div>
</div>
-
<div id="leftcontenttext" style = "width: 740px; height:180px" align="justify">
+
<div id="photos" style = "width: 300px; float:right" align="justify">
 +
<center><img style="margin-top:0px; height:250px"; padding:0;"
 +
src="<div id="photos" style = "width: 300px; float:left" align="justify">
 +
<center><img style="margin-top:0px; height:250px"; padding:0;"
 +
src="https://static.igem.org/mediawiki/2013/4/40/UCSF_LowPromoterModel.jpg"> </center>
 +
</div>
 +
"> </center>
 +
</div>
 +
 
 +
 
 +
<div id="leftcontenttext" style = "width: 300px; float:right" align="justify">
<p2><br>For the summer, we used fluorescent proteins to differentiate between our target cell strains and our unaffected cell strains. Our targeted cells will be marked with red fluorescent protein (RFP) while our unaffected cells with be marked with the fluorescent protein, citrine. Both cell strains will receive the conjugative plasmid from the donor. The gRNA-dCAS9 complex will then form and repress the production of RFP in our target cells. The RFP cell strain will no longer be able to fluoresce, since the gRNA in our conjugative plasmid only recognizes a specific site on RFP, while the citrine cell strain will be left unaffected because there is no gRNA in the conjugative plasmid that recognizes citrine. </p2>
<p2><br>For the summer, we used fluorescent proteins to differentiate between our target cell strains and our unaffected cell strains. Our targeted cells will be marked with red fluorescent protein (RFP) while our unaffected cells with be marked with the fluorescent protein, citrine. Both cell strains will receive the conjugative plasmid from the donor. The gRNA-dCAS9 complex will then form and repress the production of RFP in our target cells. The RFP cell strain will no longer be able to fluoresce, since the gRNA in our conjugative plasmid only recognizes a specific site on RFP, while the citrine cell strain will be left unaffected because there is no gRNA in the conjugative plasmid that recognizes citrine. </p2>
</div>
</div>

Revision as of 03:37, 29 October 2013

CRISPR Decision-Making Circuit

GOAL: To design a CRISPRi system with tiered responses that can be easily scaled to deploy multiple circuits within the same cell.
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 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.

Promoter Sensitivity:

To specify our outputs we want a promoter that responds to a low amount of inducer and a promoter that responds to a high amount of inducer.
">

For the summer, we used fluorescent proteins to differentiate between our target cell strains and our unaffected cell strains. Our targeted cells will be marked with red fluorescent protein (RFP) while our unaffected cells with be marked with the fluorescent protein, citrine. Both cell strains will receive the conjugative plasmid from the donor. The gRNA-dCAS9 complex will then form and repress the production of RFP in our target cells. The RFP cell strain will no longer be able to fluoresce, since the gRNA in our conjugative plasmid only recognizes a specific site on RFP, while the citrine cell strain will be left unaffected because there is no gRNA in the conjugative plasmid that recognizes citrine.

Retrieved from "http://2013.igem.org/Team:UCSF/Project/Circuit/Design1"