Team:UCSF/Project/Conjugation/Data1
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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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Revision as of 12:15, 28 October 2013
CRISPR Conjugation - Experiments and Results
Confirming Conjugation:
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%.