Team:UCSF/Project/Conjugation/Data1

From 2013.igem.org

(Difference between revisions)
(Created page with "{{Template:UCSF/MainHeader}} <html> <head> <!--CSS styles: global--> <style type="text/css"> /*** Minimal header: Thanks a lot to 2012 Calgary team for snippets of their code! C...")
Line 460: Line 460:
         myMenu.remember = false;
         myMenu.remember = false;
         myMenu.init();
         myMenu.init();
-
         myMenu.expandMenu(myMenu.submenus[0]);
+
         myMenu.expandMenu(myMenu.submenus[1]);
     };
     };
// ]]>
// ]]>
Line 501: Line 501:
<div id="box1" align="justify">
<div id="box1" align="justify">
<!------------------------------------Comtext------------------------------------->
<!------------------------------------Comtext------------------------------------->
-
<h2><center> Transmitting CRISPRi Circuits through Cell-to-Cell Conjugation </center></h2>
+
<h2><center> CRISPR Conjugation - Experiments and Results </center></h2>
<div id="leftcontenttext" style = "width: 740px;height:60px" align="justify">
<div id="leftcontenttext" style = "width: 740px;height:60px" align="justify">
-
<p1><center>GOAL: To construct a specific gene repression system using CRISPRi that can be efficiently transmitted between cells by conjugation.</center></p1>
+
<p1><center>Summary: We have successfully constructed a specific gene repression system using CRISPRi that can be efficiently transmitted between cells via conjugation.</center></p1>
</div>
</div>
-
<div id="leftcontenttext" style = "width: 740px;height:320px" align="justify">
+
<div id="leftcontenttext" style = "width: 740px;height:200px" align="justify">
-
<h3>What is conjugation? </h3>
+
<h3>Confirming Conjugation:</h3>
-
<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>
+
<p2>For conjugation to occur, the bacterial strain that passes the plasmid must have the genes necessary to build the conjugative apparatus (a pilus) between the cells. This is normally carried on a large plasmid, called the F plasmid, that itself is transferred during conjugation, but due to its large size, it is not amenable to many cloning strategies. To combat this, we chose a bacterial strain (S17-1) that has the conjugative genes integrated into the chromosome, and a small conjugative plasmid containing the origin sequence for conjugative transfer, pARO190. We were then able to clone in sequences for dCas9 and a guideRNA for the target of our knockdown – RFP.</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:200px"; padding:0;"
-
src="https://static.igem.org/mediawiki/2013/a/ac/UCSF2013-Project-Conjugation-1.png"> </center>
+
src="https://static.igem.org/mediawiki/2013/d/da/Conjugation_Plasmid.png"> </center>
</div>
</div>
-
<div id="leftcontenttext" style = "width: 740px; height:95px" align="justify">
+
<div id="leftcontenttext" style = "width: 740px; height:150px" 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>
+
<p2><br>We first needed to confirm that conjugation was possible in our experimental setup. To test this, we co-cultured the donor strain (spectinomycin resistance) containing our empty pARO190 plasmid (carbenicillin resistance) with our target strain, which has RFP  and chloramphenicol resistance intergrated into its chromosome (JM109-RFP).  At certain time points, we took a sample of the co-cultures and selected for target strain cells that have received the conjugative plasmid, which we call “transconjugates”.  On average, we obtained a conjugation efficiency of 0.4%.</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:250px"; padding:0;"
+
<center><img style="margin-top:0px; height:300px"; padding:0;"
-
src="https://static.igem.org/mediawiki/2013/8/83/Conjugation-2.png"> </center>
+
src="https://static.igem.org/mediawiki/2013/9/9f/Conjugation_Fig2_new-UCSF.png"> </center>
 +
</div>
 +
 
 +
<div id="leftcontenttext" style = "width: 640px; height:150px; margin-left:100px" align="justify">
 +
<p3>Figure 2: Effect of co-culture time on the efficiency of conjugation. Donor and target cells were diluted and mixed together so that the initial OD600 value of each co-culture was 0.05, and co-cultured in EZ-rich media at 37 ℃ under 180rpm shaking  for 2 hours, 5 hours, and 8 hours, respectively. Final cell densities were measured by plating on LB agar plates containing Spectinomycin, Chloramphenicol, and Carbenicillin + Chloramphenicol, for the selection of donor, target, and transconjugants respectively. The conjugation rate (transconjugants/target * 100%) for each experiment was 0.45%, 0.23% and 0.44%, respectively.</p2>
</div>
</div>

Revision as of 11:10, 28 October 2013

CRISPR Conjugation - Experiments and Results

Summary: We have successfully constructed a specific gene repression system using CRISPRi that can be efficiently transmitted between cells via conjugation.

Confirming Conjugation:

For conjugation to occur, the bacterial strain that passes the plasmid must have the genes necessary to build the conjugative apparatus (a pilus) between the cells. This is normally carried on a large plasmid, called the F plasmid, that itself is transferred during conjugation, but due to its large size, it is not amenable to many cloning strategies. To combat this, we chose a bacterial strain (S17-1) that has the conjugative genes integrated into the chromosome, and a small conjugative plasmid containing the origin sequence for conjugative transfer, pARO190. We were then able to clone in sequences for dCas9 and a guideRNA for the target of our knockdown – RFP.

We first needed to confirm that conjugation was possible in our experimental setup. To test this, we co-cultured the donor strain (spectinomycin resistance) containing our empty pARO190 plasmid (carbenicillin resistance) with our target strain, which has RFP and chloramphenicol resistance intergrated into its chromosome (JM109-RFP). At certain time points, we took a sample of the co-cultures and selected for target strain cells that have received the conjugative plasmid, which we call “transconjugates”. On average, we obtained a conjugation efficiency of 0.4%.
Figure 2: Effect of co-culture time on the efficiency of conjugation. Donor and target cells were diluted and mixed together so that the initial OD600 value of each co-culture was 0.05, and co-cultured in EZ-rich media at 37 ℃ under 180rpm shaking for 2 hours, 5 hours, and 8 hours, respectively. Final cell densities were measured by plating on LB agar plates containing Spectinomycin, Chloramphenicol, and Carbenicillin + Chloramphenicol, for the selection of donor, target, and transconjugants respectively. The conjugation rate (transconjugants/target * 100%) for each experiment was 0.45%, 0.23% and 0.44%, respectively.

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/Conjugation/Data1"