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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”. On average, we obtained a conjugation efficiency of 37%.
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: The dose-response curve for the CRISPRi system. JM109-RFP was transformed directly with the conjugative plasmid containing the CRISPRi system (sgRNA and dCas9 protein) for RFP. The dCas9 protein of the repression system was under control of a pTET promoter and the sgRNA of the system was under control of a constitutively expressed promoter. RFP levels were 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 direct transformation of conjugative plasmid backbone (pARO190 without sgRNA and dCas9) into JM109-RFP, and negative control was directly transform of conjugative plasmid that contains CRISPR/Cas repression system (sgRNA and dCas9 protein) for RFP into JM109 strain without any fluorescent markers. RFP levels were measured by flow cytometry and error bars showed 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 CRISPRi system transferred through conjugation from donor to recipient strain. Donor (S17-1) strain contain conjugative plasmid (sgRNA and dCas9 on pARO190 plasmid backbone) and a positive control donor strain (S17-1 strains contain only pARO190 backbone) were co-cultured with recipient strain (JM109 with RFP inserted into chromosome) for 8 hours, then diluted 100 times and start induction in new EZ-rich media. 500 ng/mL aTc were supplemented to induce the production of dCas9 protein of the CRISPRi system. Different antibiotic marker were used (Carbenicillin + Chloramphenicol, and Chloramphenicol) for the selection of recipient cells with and without conjugation. RFP fluorescence were measured by flow cytometry and error bars showed standard deviation calculated on the basis of technical replicates.
After conjugation and transfer of the conjugative plasmid, we see shifts in the populations of RFP expressing cells in those strains with our CRISPRi system compared to the control. Our data in the second trial seem to suggest that the 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.
