Thomas Eng, PhD - P. putida Transformation

Transformation via Electroporation of P. putida with Integrating or Replicating Plasmids

DNA Transformation Via Natural Bacterial Conjugation

Transformation via Electroporation of P. putida with Integrating or Replicating Plasmids

This protocol yields enough cells for transforming 10 separate transformation reactions. Scale up the volume of cells washed as necessary to transform a larger number of plasmids.

From an agar plate or overnight culture no more than ~4 days old: plate: scrape off an equivalent amount of biomass corresponding to 10 large colonies using a sterile toothpick. Resuspend in 10% glycerol. Liquid culture: Take out 500uL to 1mL of culture media of a saturated overnight culture.
Spin the cells down at 8,000 rpm for 3 minutes.
Use a sterile P1000 tip to remove 80% of the liquid. It is more important not to touch the pellet than to remove all liquid.
Add 1mL of 10% glycerol. Glycerol stored at 4C or room temperature work equally well. Gently resuspend cell pellet by pipetting up and down several times. Repeat the wash steps (steps 2-4) 2 more times for a total of 3 washes in 10% glycerol.
After washing is complete, resuspend the cell pellet in 500uL of 10% glycerol.
In sterile 1.7mL Eppendorf tubes, aliquot out 50uLs of washed cells for each separate transformation. Incubating cells at room temperature is fine – there is no need to keep cells on ice. Include a no DNA control to confirm background selection rates.
For replicating plasmids, add 100-400ng plasmid DNA to the appropriate tube.
For integrating plasmids (ie, allelic exchange vectors) use 1-2µg DNA.
Use the Bio-Rad electroporator and blue cap cuvettes (2mm gap distance) to electroporate DNA into the cells with the EC2 program. For each transformation, do the following in rapid succession:
1. Unwrap the cuvette and place into the holder.
2. Check the program is set to EC2.
3. Add ~50uL of DNA + competent cells to the cuvette.
4. Hit “START” to electroporate the sample.
5. After the sample has run, use a P20 tip fitted into a P1000 tip to take 500uL of outgrowth media to add to the cuvette. Recover the entire culture volume (approximately ~550uL). Return the sample to the same Eppendorf tube the sample came from. [The outgrowth media is preferably SOC, but TB or LB also works fine in a pinch. Outgrowth media does not contain antibiotics.]
  1. If the error message ARC appears or you hear a popping noise, change the program from EC2 to EC1 and repeat. If the program fails a second time on EC1, it is unlikely you will get any transformants. Prepare a new sample of cells + DNA, but this time reduce the amount of DNA by 2.
6. Repeat steps a-e for all remaining samples. Place the electroporated samples in the variable temperature Eppendorf tube incubator set to 30˚C and ~900 RPM. Duration of incubation is described in step 9.
Replicating plasmids: After 2-3 hours, plate 250uL of the 500uL sample on an LB + antibiotic plate. Incubate at 30C except for CRISPR based plasmids, which are incubated at 23C (room temp). A longer incubation time is ok, but if you know you need to let them incubate for 5-8 hours, drop the incubation temperature down to ~27˚C to slow the cell division rate down. With this dilution, expect to get 400-2,000 colonies after 1-2 days at 30C.

Integrating plasmids (allelic exchange plasmids): Incubate cultures for at least 8 hours, but an overnight incubation is preferable. Spin down the cell pellet (8,000 rpm 3 minutes) to remove the top ~300uL of culture. Resuspend the cell pellet in the remaining ~250uL of media. Plate the entire remaining volume on a selective plate and incubate at 30C. Expect to get around 40-100 colonies after 2-3 days at 30C.

NOTE ON PLATING:

Use a minimum number of strokes with the glass rod to spread cells. Look for the liquid media to form rivers on the plate. If you use glass beads, make sure you don’t shake the plates so hard that the glass beads simply spin along the sides of the petri dish. You are aiming to spread the culture, not to dry the plate. Excess spreading can lyse cells!!

DNA Transformation Via Natural Bacterial Conjugation

Note. If your plasmid DNA contains repetitive or duplicated elements, some conjugative hosts are not recA- endA-, and as such may exhibit greater plasmid instability. Consider directly electroporating 1-2µg plasmid DNA directly into your putida strain using the protocol in the section above. After an overnight outgrowth in non-selective media, dozens of transformants can be recovered with this alternate method. Some putida strains (such as ∆PP_1385 ttgB1) also confer sensitivity to chloramphenicol, making this method incompatible with the particular strain background since there is no way to counterselect against S17-1 without it. Electroporation of the plasmid is the recommended route for that application.

Day 0

Grow an overnight culture of E coli donor strain (S17-1) and P putida recipient strain in LB at 30C with shaking

Day 1

Take out 1mL of E coli with the plasmid. Wash your E coli cells 2x with 10% glycerol or fresh media without any antibiotic. 500uL-1mL wash volume is good, whatever is easy to handle. We don’t want to expose our Pp strains to the same antibiotics we use to propagate the plasmid yet. Resuspend the S17 with plasmid cells in ~500uL of LB media.
Prep an appropriate number of Eppendorf tubes with the Pp strains you want to conjugate with the plasmid. Add ~250uL of the Pp liquid culture to each Eppendorf tube. Once aliquoted out, spin down cells for 3min at 8k rpm.
Add 250uL of Ecoli + plasmid to the Pp cell pellet. Mix several times to resuspend.
Make 4 spots of mixed colonies on an LB plate. Don’t put cells with different plasmids or genotypes on the same plate.
Incubate at 30˚C for 24-48h in the dark

Day 2

On a LB CMR + KAN/GNT plate, use a small amount of cells (5uL) from each spot to streak to singles. The rate of KAN/GNT CMR cells will be low so make sure there is ample space to pick colonies from the 1st and 2nd toothpick areas. Divide the plate into quadrants for streaking

Day 3, and Day 4

Usually we see single colonies within a day or 2 of streaking. Pick 4 well formed and distinct single colonies (one from each quadrant) and restreak again on the same type of selective media for additional (and required) purification. Quadrants on a plate work well, this time 4 toothpicks work well.

Part 2: Double Crossover Selection

Day 5

From each quadrant, pick a single colony. Innoculate the single colony into LB media (no antibiotics!!!) either liquid LB is fine or on a solid agar LB plate - you can get to a saturated liquid LB culture faster if you’re in a rush
Incubate at 30C for at least 6 hours with shaking (cells should hit log phase pretty fast from a single colony), overnight is fine
Take ~5-10 uL of culture and spot onto a LB SUC plate (or a small amount of biomass equivalent to a large colony from the LB agar plate). Streak to singles. Give lots of space in the first and second toothpick area to pick singles. Divide the plate into 2 quadrants so you have plenty of space. Results are better if the SUC plates are left at 23C room temp

Day 6

Pick ~40 sucrose resistant clones and patch onto LB KAN/GNT and LB SUC plates. Put a big X over patches which are KAN/GNT resistant (these are parental colonies we don’t want)

Day 7

Identify WT revertants and deletions by colony PCR (outside/outside, at least one inside/outside flanking pair). Pick a small amount of colony biomass and boil in 20mM NaOH for 40 minutes. Then use NEB 2x OneTaq MM to screen. A 3 (three, not thirty) second anneal time works well to reduce background amplicons.

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