TITLE: Triparental mating with pSEVA protocol
AUTHORS: Laura Gómez

[DESCRIPTION]
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.


[STEPS]
SECTION: Insertion of he suicide plasmid by three partner conjugation
1. 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) 
 
 

SECTION: Insertion of he suicide plasmid by three partner conjugation
2. 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).
SECTION: Insertion of he suicide plasmid by three partner conjugation
3. Leave the plate open at the flame until the spots get dry. Incubate at 37 °C 120 min, facing up.

SECTION: Insertion of he suicide plasmid by three partner conjugation
4. 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 1440 min 37 °C facing up.

SECTION: Insertion of he suicide plasmid by three partner conjugation
5. Collect the 4 patches using a sterile loop and resuspend each of them in 1 ml of LB in an eppendorf.
SECTION: Insertion of he suicide plasmid by three partner conjugation
6. Plate 100 µl of each tube in LB plates supplemented with Gm + Sm.
SECTION: Insertion of he suicide plasmid by three partner conjugation
7. Centrifuge the rest of the D+H+R tube at 2000 x g, 2 min to pellet the cells, resuspend in 100 µl and plate as well.

SECTION: Insertion of he suicide plasmid by three partner conjugation
8. Incubate the plates 1440 min at 37°C.

SECTION: Second recombination for a scarless genomic modification
9. 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 360 min for the induction of the I-SceI endonuclease of the pACBSR plasmid.

 


SECTION: Second recombination for a scarless genomic modification
10. Insert the inoculation loop in the culture and streak on LB+Sm plates to obtain individual colonies. Incubate the plates 1440 min at 37°C.
SECTION: Second recombination for a scarless genomic modification
11. The next day, pick some colonies and patch them on a LB+Sm plate and on a LB+Sm+Gm plate. Incubate the plates 1440 min at 37°C.

 

SECTION: Second recombination for a scarless genomic modification
12. 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.
SECTION: Second recombination for a scarless genomic modification
13. 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.