Team:UCSF/Project/Circuit/Design1
From 2013.igem.org
(Difference between revisions)
Line 532: | Line 532: | ||
</div> | </div> | ||
+ | <div id="leftcontenttext" style = "width: 740px; float:left" align="justify"> | ||
+ | <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> | ||
+ | </div> | ||
+ | <div id="leftcontenttext" style = "width: 740px; height:95px" align="justify"> | ||
+ | <h3>CRISPRi Circuit Design:</h3> | ||
+ | <p2>fluorescent protein and a gRNA. The gRNA will then form a complex with dCAS9, which is on a separate plasmid, and block the production of the opposite fluorescent protein. Only one fluorescent protein should be produced as our output. There are two scenarios that our circuit is capable of producing. </p2> | ||
+ | </div> | ||
- | <div id=" | + | <div id="photos" style = "width: 740px; float:left" align="justify"> |
- | < | + | <center><img style="margin-top:0px; height:250px"; padding:0;" |
+ | src="https://static.igem.org/mediawiki/2013/4/4a/Synthetic_Circuit_Picture.jpg"> </center> | ||
</div> | </div> | ||
- | <div id=" | + | <div id="leftcontenttext" style = "width: 740px; float:left" align="justify"> |
- | < | + | <p2><br>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).</p2> |
- | + | ||
</div> | </div> | ||
+ | |||
+ | <div id="photos" style = "width: 740px; float:left" align="justify"> | ||
+ | <center><img style="margin-top:0px; height:250px"; padding:0;" | ||
+ | src="https://static.igem.org/mediawiki/2013/8/88/GFP_output.jpg"> </center> | ||
+ | </div> | ||
+ | |||
+ | <div id="photos" style = "width: 740px; float:left" align="justify"> | ||
+ | <center><img style="margin-top:0px; height:250px"; padding:0;" | ||
+ | src="https://static.igem.org/mediawiki/2013/d/d5/RFP_output.jpg"> </center> | ||
+ | </div> | ||
+ | |||
+ | <div id="leftcontenttext" style = "width: 740px; height:95px" align="justify"> | ||
+ | <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> | ||
+ | <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>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>. | ||
+ | </p2> | ||
+ | </div> | ||
+ | |||
+ | <div id="leftcontenttext" style = "width: 740px; height:95px" align="justify"> | ||
+ | <h3>Using CRISPR to Create Scalable Circuits:</h3> | ||
+ | <p2>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. </p2> | ||
+ | |||
+ | <div id="photos" style = "width: 740px; float:left" align="justify"> | ||
+ | <center><img style="margin-top:0px; height:250px"; padding:0;" | ||
+ | src="https://static.igem.org/mediawiki/2013/8/8b/Scalable_Circuit.jpg"> </center> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
<!------------------------------------End Comtext-------------------------------------> | <!------------------------------------End Comtext-------------------------------------> |
Revision as of 03:43, 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.