Triparental mating with pSEVA protocol
Laura Gómez
Disclaimer
Abstract
This process involves bacterial conjugation, where a conjugative plasmid found in one bacterial strain facilitates the transfer of a mobilizable plasmid from a second bacterial strain to a third bacterial strain.
In the method from our lab, CC118λpir containing a pSEVA plasmid works as the donor, the E. coli 1047 pRK2013 strand is the helper, and the receiver is a C. rodentium strand.
Before start
Steps
Insertion of he suicide plasmid by three partner conjugation
Inoculate cultures of strains: DONOR CC118λpir pSEVA Gm (10 μg/ml) HELPER E. coli 1047 pRK2013 Km (50 μg/ml) RECEIVER C. rodentium pACBSR Sm (50 μg/ml)
Place 20 µl spots of the helper and donor strains onto an LB plate (no antibiotics) and an additional spot of 20 µl of the helper on top of 20 µl of the donor (D+H) .
Leave the plate open at the flame until the spots get dry. Incubate at 37°C 2h 0m 0s, facing up.
Add 40 µl of the receiver strain on top of the D+H spot (D+H+R) and an additional 20 µl spot of this strain alone. Wait for the spot to get dry and incubate for 24h 0m 0s 37°C facing up.
Collect the 4 patches using a sterile loop and resuspend each of them in 1 ml of LB in an eppendorf.
Plate 100 µl of each tube in LB plates supplemented with Gm + Sm .
Centrifuge the rest of the D+H+R tube at 2000x g to pellet the cells, resuspend in 100 µl and plate as well.
Incubate the plates 24h 0m 0s at 37°C.
Second recombination for a scarless genomic modification
Pick two colonies of the D+H+R plate and grow them in LB+Sm + L-arabinose broth at 0.4% for a minimum of 6h 0m 0s for the induction of the I-SceI endonuclease of the pACBSR plasmid.
Insert the inoculation loop in the culture and streak on LB+Sm plates to obtain individual colonies. Incubate the plates 24h 0m 0s at 37°C.
The next day, pick some colonies and patch them on a LB+Sm plate and on a LB+Sm+Gm plate. Incubate the plates 24h 0m 0s at 37°C.
Analyze by PCR and gel electrophoresis those colonies which have grown on LB+Sm but not on LB+Sm+Gm to differentiate the modified colonies from the ones which have reverted to the Wild-Type genotype. NOTE : The primers should hybridise outside of the homology regions selected. Upon analysis of 10 colonies you should get a about 50% of modified colonies.
NOTE : To remove the pACBSR plasmid, make 8-9 passes of the strain without Sm in liquid LB. Plate the last culture in LB plates and patch individual colonies the following day on LB and LB+Sm plates to select those sensitive to the antibiotic.
