Team:UCSF/Project/Conjugation/Data1

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CRISPRi Conjugation Project Design Data

CRISPRi Circuit Circuit Design Promoter Engineering Data

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 efficiency for each experiment was 0.45%, 0.23% and 0.44%, respectively.

Knock-down of RFP with CRISPRi:

Next, we needed to confirm that the CRISPRi system we designed could sufficiently repress RFP expression in our target strain. By directly transforming our engineered conjugative plasmid (which contains our CRISPRi system) into our target cells, we were able to measure RFP fluorescence after induction of the conjugative plasmid. We were able to observe significant knock-down of RFP expression in the cell containing our CRISPRi system.

Figure 3: The efficiency of our CRISPRi system. The positive control is a direct transformation of the conjugative plasmid backbone (pARO190 without gRNA and dCas9) into our recipient cells. RFP fluorescence is measured via plate reader and error bars show standard deviation calculated on the basis of technical replicates.

Figure 4: The dose-response curve for our CRISPRi (dCas9 and gRNA) system. RFP fluorescence was measured under different aTc concentrations (0, 0.625, 1, 6.25, 9.375, 12.5, 18.75, 25, 37.5, 50, 250, 500 ng/mL aTc). Positive control was a direct transformation of the conjugative plasmid backbone (pARO190 without gRNA and dCas9) into our target cells, and negative control was directly transform of conjugative plasmid into JM109 strain without any fluorescent markers. RFP fluorescence was measured via flow cytometry and error bars show standard deviation calculated on the basis of technical replicates.

Conjugation of the CRISPRi System into Target Cells:

After demonstrating that our delivery (conjugation) and repression systems (CRISPRi system) are both functional in our assays, we moved on to test whether or not we can do both. That is, if it is possible to induce knock-down of a specific gene by transferring specific instructions from one cell to another via conjugation. We co-cultured our donor strain containing the CRISPRi system with the JM-109 target strain for eight hours. The cultures were then diluted and selected for transconjugates by antibiotic resistance (cam and carb) and then induced with aTc to express dCas9 on the conjugative plasmid. Using FACS, we measured the RFP output post-conjugation at various time points post-induction.

Figure 5: Conjugation of the CRISPRi System into Target Cells. Our donor (S17-1) strain, which contains our conjugative plasmid (gRNA and dCas9 on pARO190 plasmid backbone) and a positive control donor strain (S17-1 containing only pARO190 backbone) were co-cultured separately with our target strain (JM109 with RFP inserted into its chromosome) for 8 hours. The co-cultures were then diluted 100 times and inducted with 500ng/mL aTc in new EZ-rich media. Different antibiotic markers were used (Carbenicillin + Chloramphenicol, and Chloramphenicol) to select for target cells with and without the conjugative plasmid. The histograms show RFP fluorescence for each sample, which was measured via flow cytometry.

Figure 6: Conjugation of the CRISPRi System into Target Cells. Our donor (S17-1) strain, which contains our conjugative plasmid (gRNA and dCas9 on pARO190 plasmid backbone) and a positive control donor strain (S17-1 containing only pARO190 backbone) were co-cultured separately with our target strain (JM109 with RFP inserted into its chromosome) for 8 hours. The co-cultures were then diluted 100 times and inducted with 500ng/mL aTc in new EZ-rich media. Different antibiotic markers were used (Carbenicillin + Chloramphenicol, and Chloramphenicol) to select for target cells with and without the conjugative plasmid. RFP fluorescence was measured via flow cytometry and error bars show standard deviation calculated on the basis of technical replicates.

After the transfer of the conjugative plasmid, there is a decrease in RFP expression in strains that have received the CRISPRi system compared to the control (pARO190 backbone), which shows no significant change. Our data in the second trial seem to suggest that dCas9 expression is somewhat leaky behind our pTET promoter, for there is expression of dCas9 without induction. Since our guideRNA is constitutively expressed, this results in a small amount of specific targeting, and therefore RFP repression, without induction. It also appears that overexpression of the dCas9 protein could be somewhat toxic to the cells, so we are currently working on modifying the promoter to achieve a tighter regulation of dCas9 expression.

Specificity of targeting through CRISPRi Conjugation

To test the specificity of our system, we obtained a strain of E. coli (kindly provided by Stanley Qi, UCSF) containing both RFP and GFP integrated into its chromosome. We then conjugated our donor strain with this new recipient strain and measured RFP and GFP fluorescence. As can be seen in the graphs below, RFP fluorescence decreased only when the plasmid containing dCas9 and RFP-gRNA were conjugated. GFP fluorescence was not affected in any of the conditions, showing that the dCas9-gRNA complex transferred into cells via conjugation is highly specific.

We have shown that a specific gene repression system using CRISPRi can be efficiently transmitted between cells by conjugation.

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        <div class="collapsed">
          <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>
        </div>
        <div class="collapsed">
          <span>CRISPRi Circuit</span>
-         <a href="/Team:UCSF/Project/Circuit/Design">Design</a>
+         <a href="/Team:UCSF/Project/Circuit/Design">Circuit Design</a>
+        <a href="/Team:UCSF/Project/Circuit/Design">Promoter Engineering</a>
          <a href="/Team:UCSF/Project/Circuit/Data">Data</a>
        </div>
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 <!------------------------------------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:50px" 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 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 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 id="leftcontenttext" style = "width: 740px; height:95px" align="justify">
+<div id="leftcontenttext" style = "width: 740px; height:155px" 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 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: 740px; height:180px" align="justify">
+<div id="leftcontenttext" style = "width: 600px; height:150px; margin-left:80px" 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>
+<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 efficiency for each experiment was 0.45%, 0.23% and 0.44%, respectively.</p2>
+</div>
+<div id="leftcontenttext" style = "width: 740px; height:140px" align="justify">
+<h3>Knock-down of RFP with CRISPRi:</h3>
+<p2>Next, we needed to confirm that the CRISPRi system we designed could sufficiently repress RFP expression in our target strain. By directly transforming our engineered conjugative plasmid (which contains our CRISPRi system) into our target cells, we were able to measure RFP fluorescence after induction of the conjugative plasmid. We were able to observe significant knock-down of RFP expression in the cell containing our CRISPRi system.</p2>
 </div>
 <div id="photos" style = "width: 740px" align="justify">
-<center><img style="margin-top:0px; height:350px"; padding:0;"
+<center><img style="margin-top:0px; height:300px"; padding:0;"
-src="https://static.igem.org/mediawiki/2013/7/70/Target_and_unaffected_cells.jpg"> </center>
+src="https://static.igem.org/mediawiki/2013/f/f8/Conjugation_Fig3_new_UCSF.png"> </center>
 </div>
+<div id="leftcontenttext" style = "width: 550px; height:90px; margin-left:120px" align="justify">
+<p3>Figure 3: The efficiency of our CRISPRi system. The positive control is a direct transformation of the conjugative plasmid backbone (pARO190 without gRNA and dCas9) into our recipient cells. RFP fluorescence is measured via plate reader and error bars show standard deviation calculated on the basis of technical replicates.</p2>
+</div>
+<div id="photos" style = "width: 740px" align="justify">
+<center><img style="margin-top:0px; height:300px"; padding:0;"
+src="https://static.igem.org/mediawiki/igem.org/5/52/Direct_Transformation_Concentrations.jpg"> </center>
+</div>
+<div id="leftcontenttext" style = "width: 600px; height:150px; margin-left:80px" align="justify">
+<p3>Figure 4: The dose-response curve for our CRISPRi (dCas9 and gRNA) system. RFP fluorescence was measured under different aTc concentrations (0, 0.625, 1, 6.25, 9.375, 12.5, 18.75, 25, 37.5, 50, 250, 500 ng/mL aTc). Positive control was a direct transformation of the conjugative plasmid backbone (pARO190 without gRNA and dCas9) into our target cells, and negative control was directly transform of conjugative plasmid into JM109 strain without any fluorescent markers. RFP fluorescence was measured via flow cytometry and error bars show standard deviation calculated on the basis of technical replicates.</p2>
+</div>
+<div id="leftcontenttext" style = "width: 740px; height:195px" align="justify">
+<h3>Conjugation of the CRISPRi System into Target Cells:</h3>
+<p2>After demonstrating that our delivery (conjugation) and repression systems (CRISPRi system) are both functional in our assays, we moved on to test whether or not we can do both. That is, if it is possible to induce knock-down of a specific gene by transferring specific instructions from one cell to another via conjugation.</p2>
+<p2>We co-cultured our donor strain containing the CRISPRi system with the JM-109 target strain for eight hours. The cultures were then diluted and selected for transconjugates by antibiotic resistance (cam and carb) and then induced with aTc to express dCas9 on the conjugative plasmid. Using FACS, we measured the RFP output post-conjugation at various time points post-induction.</p2>
+</div>
+<div id="photos" style = "width: 740px" align="justify">
+<center><img style="margin-top:0px; height:300px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/b/b5/Conjugation_Fig5_new_UCSF.png"> </center>
+</div>
+<div id="leftcontenttext" style = "width: 600px; height:160px; margin-left:80px" align="justify">
+<p3>Figure 5: Conjugation of the CRISPRi System into Target Cells. Our donor (S17-1) strain, which contains our conjugative plasmid (gRNA and dCas9 on pARO190 plasmid backbone) and a positive control donor strain (S17-1 containing only pARO190 backbone) were co-cultured separately with our target strain (JM109 with RFP inserted into its chromosome) for 8 hours. The co-cultures were then diluted 100 times and inducted with 500ng/mL aTc in new EZ-rich media. Different antibiotic markers were used (Carbenicillin + Chloramphenicol, and Chloramphenicol) to select for target cells with and without the conjugative plasmid. The histograms show RFP fluorescence for each sample, which was measured via flow cytometry.</p2>
+</div>
+<div id="photos" style = "width: 740px" align="justify">
+<center><img style="margin-top:0px; height:300px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/a/a5/Conjugation_Fig6_new_UCSF.png"> </center>
+</div>
+<div id="leftcontenttext" style = "width: 600px; height:160px; margin-left:80px" align="justify">
+<p3>Figure 6: Conjugation of the CRISPRi System into Target Cells. Our donor (S17-1) strain, which contains our conjugative plasmid (gRNA and dCas9 on pARO190 plasmid backbone) and a positive control donor strain (S17-1 containing only pARO190 backbone) were co-cultured separately with our target strain (JM109 with RFP inserted into its chromosome) for 8 hours. The co-cultures were then diluted 100 times and inducted with 500ng/mL aTc in new EZ-rich media. Different antibiotic markers were used (Carbenicillin + Chloramphenicol, and Chloramphenicol) to select for target cells with and without the conjugative plasmid. RFP fluorescence was measured via flow cytometry and error bars show standard deviation calculated on the basis of technical replicates.</p2>
+</div>
+<div id="leftcontenttext" style = "width: 740px; height:165px" align="justify">
+<p2>After the transfer of the conjugative plasmid, there is a decrease in RFP expression in strains that have received the CRISPRi system compared to the control (pARO190 backbone), which shows no significant change. Our data in the second trial seem to suggest that dCas9 expression is somewhat leaky behind our pTET promoter, for there is expression of dCas9 without induction. Since our guideRNA is constitutively expressed, this results in a small amount of specific targeting, and therefore RFP repression, without induction. It also appears that overexpression of the dCas9 protein could be somewhat toxic to the cells, so we are currently working on modifying the promoter to achieve a tighter regulation of dCas9 expression.</p2>
+</div>
+<div id="leftcontenttext" style = "width: 740px; height:195px" align="justify">
+<h3>Specificity of targeting through CRISPRi Conjugation</h3>
+<p2>To test the specificity of our system, we obtained a strain of E. coli (kindly provided by Stanley Qi, UCSF) containing both RFP and GFP integrated into its chromosome. We then conjugated our donor strain with this new recipient strain and measured RFP and GFP fluorescence. As can be seen in the graphs below, RFP fluorescence decreased only when the plasmid containing dCas9 and RFP-gRNA were conjugated. GFP fluorescence was not affected in any of the conditions, showing that the dCas9-gRNA complex transferred into cells via conjugation is highly specific.</p2>
+</div>
+<div id="photos" style = "width: 740px" align="justify">
+<center><img style="margin-top:0px; height:300px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/1/1f/Conjugation-GFP-RFP-UCSF.png"> </center>
+</div>
+<div id="photos" style = "width: 740px" align="justify">
+<center><img style="margin-top:0px; height:300px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/f/f9/Conjugation-GFP-RFP-UCSF2.PNG"> </center>
+</div>
+<div id="leftcontenttext" style = "width: 740px; height:145px" align="justify">
+<p1><center>We have shown that a specific gene repression system using CRISPRi can be efficiently transmitted between cells by conjugation. </center></p1>
+</div>
 <!------------------------------------End Comtext------------------------------------->