ssDNA Oligo Recombineering in Pseudomonas putida KT2440

This detailed method expands on the recombineering method described in Czajka et al (Metabolic Engineering Communications doi: 10.1016/j.mec.2022.e00206). The use of Cpf1 for selection instead of Cas9 is the main difference between what we do compared to what Aparicio et al describe (doi.org/10.1111/1751-7915.13453). There are many advantages to this method since it uses cheap, easily available oligonucleotides; The tedious creation of dsDNA PCR fragments that contain antibiotic cassettes with homology ears has many drawbacks both in generation and strain creation. For example there is no "scar" DNA left behind in the mutant strain, no potential polar effects from the new promoter sequence. With such ease in creating mutant strains it leaves more time to build plasmid-based constructs for complementation and genetic analysis, the other half of determining mutant gene function. --T.E., Nov 2022 

1. From a single colony on a LB GNT plate harboring a recombineering plasmid, pick a single large colony and inoculate into a 5mL culture tube. Put the culture at 30˚C overnight with shaking.

2. In the morning, back dilute the culture into fresh LB GNT media - take 2.5mL out and add it to 25mL LB GNT in a 250mL baffled shake flask.

3. Allow cells to grow from step 2 for an additional 1 hour at 30C shaking with 200RPM shaking after back diluting into the 250mL shake flask.

4. Add 3-methyl-benzoate (Sigma: T36609; M-Toluic Acid 99% purity) to the culture for a final concentration of 1mM 3MB. Incubate for an additional 30 minutes at 30 ˚C with shaking.

5. After 30 minutes of 3MB induction, cells are ready for recombineering via electroporation. Decant volume into a 50mL falcon tube. Spin cells down at 4˚C in the big centrifuge: 5 minutes at 4500rpm. Label the sides of the falcon tubes to prevent strain mixups.

6. Discard LB GNT media; add 500uL 10% glycerol to cell pellet, vortex cell pellet to resuspend.

7. Recover cells and transfer to 1.5mL centrifuge tube, volume will be around 1mL

8. Spin down at 8000rpm for 3 minutes.

9. Wash 2 more times in 10% cold glycerol stored in the fridge, cell pellet will become loose and fluffy with each repeated wash

10. Final volume: Resuspend cells in 1mL 10% glycerol - don't want to use too many cells in each electroporation, but adjust volume if the cell pellet seems small

11. Aliquot 50 µL cells into each tube for recombineering reaction.

12. Add 1 µL of 100 µM oligo stock and 1 µL (~25-50ng) CRISPR plasmid.

13. Use Biorad Program EC-2 with the electroporator by the window in SYNBIO-1. Recover cells with a P20 tip fitted into a P1000 tip by resuspending cells in 600uL TB media. Do this fast and within 30 seconds of the pulse completing.

14. Outgrow cells in a Thermo Thermomixer set to 1200 rpm and 30˚C for 3-4 hours. We don't exceed 6 hours. 

15.  Plate 250uL on LBK and incubate plates at 30 ˚C after dry. 

16. If successful, clones will be pinpoint after 1 day but colonies will be clearly visible after 2 days. Genotype using outside-outside and inside-inside primers (no shared homology to the recombineering oligo is allowed). For 1uL of a 100uM oligo stock and 50ng of plasmid, between 50-150 colonies should appear on the plate after 2 days. Background KAN+ rates are around 0-20 colonies (this is elevated compared to strains without recombineering plasmid pTE452). Screen 10-30 clones per genotype. Positive clone rate observed varies between 45% and 100% positive for >45 different loci. No allele heterozygosity was observed when screening directly from the transformation plate. Occasionally no recombinants are recovered (n=1 out of 50+ trials). We don’t know why. We suggest repeating the recombineering run with  more oligo. 

17. To prepare cells for the next round of modification, purify clones by restreaking one additional time on LBKAN. Then passage cells in LB (without antibiotic, liquid cultures appear to work better for plasmid curing). From the LB plate, streak colonies to singles. Pick single colonies and patch on LBKAN, LB, and LB GNT. Identify KanS GntS clones to transform with pTE452. Restart on step 1 of this protocol.