Team:UCSF/Project/Conjugation/Data1

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         <span>CRISPRi Conjugation</span>         

         <span>CRISPRi Conjugation</span>         

         <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Data1">Data</a>

         <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Data1">Data</a>

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         <span>CRISPRi Circuit</span>

         <span>CRISPRi Circuit</span>

         <a href="/Team:UCSF/Project/Circuit/Data">Data</a>

         <a href="/Team:UCSF/Project/Circuit/Data">Data</a>

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<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>

<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>

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<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>

<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>

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<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>

<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>

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<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>

<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>

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<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>

<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>

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