Team:UCSF/Project/Conjugation/Data1

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CRISPR Conjugation - Experiments and Results

Summary: We have successfully constructed a specific gene repression system using CRISPRi that can be efficiently transmitted between cells via conjugation.

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.

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%.
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.

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.

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