TITLE: Transforming the marine bacterium Ruegeria pomeroyi using tri-parental mating
AUTHORS: Cherry Gao, Christopher R. Reisch, Mary Ann Moran, Roman Stocker

[DESCRIPTION]
This protocol describes a tri-parental mating method for transforming the model marine bacterium Ruegeria pomeroyi DSS-3 with a desired recombinant plasmid. This transformation method is reliable, easy-to-use, and does not require specialized equipment. Using this method, we have successfully transformed R. pomeroyi with large plasmids (up to 14.833 kb) which enabled the construction of the first fluorescent reporter strains of R. pomeroyi (described in doi.org/10.1038/s41467-020-15693-z). 

[BEFORE_START]
Before using this protocol, the user should have designed and built the plasmid desired for transforming into R. pomeroyi. The authors have built and successfully transformed reporter plasmids on vector backbones pBBR1MCS-KanR (GenBank U23751) and pRK415 (GenBank EF437940). Furthermore, if the desired plasmid is small (empirically, the authors suggest less than 11 kb), electroporation may be a faster method for transforming R. pomeroyi. Recommendations for plasmid design and electroporation method for R. pomeroyi are available in detail in Cherry Gao’s PhD Thesis (https://hdl.handle.net/1721.1/129213).

[STEPS]
SECTION: Execution
1. Prepare    5 mL of the three bacterial strains for tri-parental mating:

(1) wildtype R. pomeroyi (1/2 YTSS medium, 30 °C  with shaking);

(2) helper E. coli containing the pRK600 plasmid (LB medium amended with 15 µg/mL , 37 °C  with shaking)

 (3) donor E. coli containing the constructed plasmid (LB medium amended with 50 µg/mL , 37 °C  with shaking).

SECTION: Execution
2. After overnight growth, wash the two E. coli cultures twice in 1/2 YTSS medium to eliminate antibiotics.
SECTION: Execution
3. To wash, aliquot 1 mL into Eppendorf tubes, pellet the bacterial cells through gentle centrifugation 3000 rpm, 5 min (on a tabletop centrifuge), discard the supernatant, and resuspend in 1 mL.

SECTION: Execution
4. Pellet the E. coli cells again through centrifugation, discard the supernatant, then resuspend in 1 mL.

SECTION: Execution
5. In a clean Eppendorf tube, combine the following: 2 mL, 200 µL and 200 µL.

SECTION: Execution
6. Gently centrifuge this mixture to pellet the bacterial cells. Discard the supernatant and resuspend in 100 µL.

SECTION: Execution
7. Deposit 50 µL onto the center of a pre-warmed 1/2 YTSS plate. Place the lid onto the plate.

SECTION: Execution
8. Incubate the plate (lid-side up)    at 30 °C  to allow mating to occur.

SECTION: Execution
9. For selection of plasmid-containing R. pomeroyi, pick up most of the biomass (now dry) that is at the center of the mating plate with a sterile L-shaped cell spreader, and evenly spread this biomass onto a 1/2 YTSS plate amended with 50 µg/mL and 50 µg/mL. 
 


SECTION: Execution
10. Incubate the selection plate (lid-side down) at 30 °C  until colonies of transformed R. pomeroyi form. Typically, small colonies become visible within 48–72 hours. Due to the presence of potassium tellurite, R. pomeroyi colonies appear dark brown.

SECTION: Execution
11. To avoid evaporation during this long incubation time, secure the lid to the plate with Parafilm.
SECTION: Execution
12. Confirm successfully transformed R. pomeroyi through colony PCR and sequencing. If possible, 16S sequencing to confirm the identity of R. pomeroyi is recommended.