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Team:UCSF/Project/Conjugation/Data
From 2013.igem.org
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.
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”.
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.
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.
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 was measured by directly transforming the conjugative plasmid that contains our CRISPRi system (gRNA and dCas9 protein) into our recipient cells (JM109 with RFP inserted into its genome). The dCas9 protein is under the control of a pTET promoter and the gRNA is under the control of a constitutive promoter. 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: Figure 4: The dose-response curve for our CRISPRi (dCas9 and gRNA) system. The conjugative plasmid containing our CRISPRi system was directly transformed into our target strain (JM109-RFP) and 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). Our positive control was a direct transformation of the conjugative plasmid backbone (pARO190 without gRNA and dCas9) into our target strain, and our negative control was directly transform of conjugative plasmid that contains CRISPR/Cas repression system (gRNA and dCas9 protein) for RFP 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.
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: The effectiveness of our CRISPRi system transferred via conjugation from donor to target strain. 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: The effectiveness of our CRISPRi system transferred via conjugation from donor to target strain. 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.
