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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 the conjugation. Because of its large size, it is not amenable to many cloning strategies. We instead chose a bacterial strain with the conjugative genes integrated into the chromosome, strain S17-1, and a small conjugative plasmid containing the origin sequence for conjugative transfer, pARO190. We were able to then 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. Briefly, we co-cultured the donor strain (spectinomycin resistance) containing our empty pARO190 plasmid (carbenicillin resistance) with a recipient strain in which we integrated both RFP and chloramphenicol resistance into the chromosome (JM109-RFP). At certain time points, we took a sample of the co-cultures and selected for only those cells that were both recipient strains (chloramphenicol) and now had the antibiotic resistance from the conjugative plasmid (carbenicillin). These we called “transconjugates”. On average, we obtained a conjugation efficiency of 37%.
Figure 2: Effect of co-culture time on the efficiency of conjugation. Donor and recipient cells were diluted and mixed together so that the initial OD600 value of each co-culture were 0.05 and then co-cultured in EZ-rich media for 2 hours, 5 hours, and 8 hours, respectively, under 180 rpm shaking in a 37 ℃ shaker. The final cell density was measured by plating on LB agar plates containing Spectinomycin, Chloramphenicol, and Carbenicillin + Chloramphenicol, for the selection of donor, recipient, and transconjugants (recipient received the conjugation plasmid) respectively. The conjugation rate (transconjugants/recipient * 100%) for each experiment was 0.45%, 0.23% and 0.44%, respectively.
Knock-down of RFP with CRISPRi:
Next, we confirmed that the CRISPR system we designed could repress RFP expression in our recipient strain. Using direct transformation of our engineered conjugative plasmid pARO190 containing both dCas9 and a guideRNA for RFP, we measured the fluorescence output of RFP after induction of the conjugative plasmid. We were able to observe significant knock-down of RFP expression in the cell containing our CRISPR system.
Next, we confirmed that the CRISPR system we designed could repress RFP expression in our recipient strain. Using direct transformation of our engineered conjugative plasmid pARO190 containing both dCas9 and a guideRNA for RFP, we measured the fluorescence output of RFP after induction of the conjugative plasmid. We were able to observe significant knock-down of RFP expression in the cell containing our CRISPR system.
Figure 3: The efficiency of CRISPRi system measured by directly transforming the conjugative plasmid that contains CRISPRi system (sgRNA and dCas9 protein) for RFP into JM109 strain that has RFP inserted into its chromosome (JM109-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. Positive control was direct transformation of conjugative plasmid backbone (pARO190 without sgRNA and dCas9) into JM109-RFP and negative control was direct transformation 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.
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:
With both the delivery system and the CRISPR repression functional in our assay, we moved to test both pieces together and 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 recipient strain for eight hours. The cultures were 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.
With both the delivery system and the CRISPR repression functional in our assay, we moved to test both pieces together and 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 recipient strain for eight hours. The cultures were 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.
Figures 5 and 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. The histogram displayed RFP fluorescence for each sample measured by flow cytometry.
Figures 7 and 8: 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 control. Our data in the second trial seem to suggest that the dCas9 expression is somewhat leaky behind our pTET promoter, showing low levels of expression without induction. Since our guideRNA is constitutively produced, this results in a small amount of specific targeting, and therefore RPF repression, without induction. It also appears as if overexpression of the dCas9 protein could be somewhat toxic to the cells, so we are currently working to modify the promoter to achieve more tight regulation of dCas9 expression.
