Team:UCSF/Project/Circuit/Design1

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         <span>CRISPRi Conjugation</span>         
         <span>CRISPRi Conjugation</span>         
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         <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Design1">Design</a>
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         <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>
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         <span>CRISPRi Circuit</span>
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         <a href="/Team:UCSF/Project/Circuit/Design">Design</a>
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         <a href="/Team:UCSF/Project/Circuit/Design1">Circuit Design</a>
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         <a href="/Team:UCSF/Project/Circuit/Data">Data</a>
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        <a https://2013.igem.org/Team:UCSF/Project/Conjugation/Promoter1">Promoter Engineering</a>
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         <a href="/Team:UCSF/Project/Circuit/Data1">Data</a>
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<h2><center> Transmitting CRISPRi Circuits through Cell-to-Cell Conjugation </center></h2>
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<h2><center> CRISPR Decision-Making Circuit </center></h2>
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<p1><center>GOAL: To construct a specific gene repression system using CRISPRi that can be efficiently transmitted between cells by conjugation.</center></p1>
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<p1><center>GOAL: To design a CRISPRi system with tiered responses that can be easily scaled to deploy multiple circuits within the same cell. </center></p1>
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<h3>What is conjugation? </h3>
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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>
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<p2>In nature, bacterial strains rarely exist as distinct populations. Instead, they are almost always found in mixed populations where they compete for resources. Conjugation is a naturally occurring process in bacteria that allows genetic material to be transferred between populations of bacterial cells. This process promotes gene diversity, and in certain situations, provides a competitive advantage for the recipient cell.<br><br></p2>
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<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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<h3>Combining CRISPRi and Conjugation</h3>
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<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>
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<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>
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<h3>Promoter Sensitivity:</h3>
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<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>
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<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>
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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>
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<h3>CRISPRi Circuit Design:</h3>
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<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>
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<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>
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src="https://static.igem.org/mediawiki/2013/8/88/GFP_output.jpg"> </center>
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<h3>Design Requirements:</h3>
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<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>
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<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>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.
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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>.
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<h3>Using CRISPR to Create Scalable Circuits:</h3>
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<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>
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Latest revision as of 03:55, 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.

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:

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.

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:

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. 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:

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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