FLP FRT Recombination

Protocol Reference: Timescale: 3 - 4 days

Brief Description

FLP-FRT recombination uses a temperature induction of the pCP20 plasmid to excise DNA that is flanked between two FRT sequences. While technically simple, the whole process takes 3 - 4 days depending on when you start inducing the FLP recombination. The most annoying step is screening through potential recombinants as the efficiency is strain-dependent.

Recombination will only occur between FRT loci, which are identical with the sequence

FRT (5’ → 3’): GAAGTTCCTATTCtctagaaaGAATAGGAACTTC

Upon recombination, a “scar sequence” will be left behind.

Original Reference

Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant

Step 1: Transformation of pCP20

Materials & Equipment

Material	Amount	Function
Electrocompetent cells of recipient (see Electrocompetent cell preparation protocol)	≥ 50 µL per recipient	Strain to which pCP20 will be transformed and FRT-flanked cassette will be removed
LB Medium	≥ 1 mL per recipient	Rich growth medium for recovery of pCP20 transformation and for executing FLP recombination.
Electroporator	—	For electroporating pCP20 into the recipient
1cm gap cuvette	1 per recipient	for electroporation
Waterbath shaker	1	For recovery of transformed strain
Glass beads / Spreader	—	For spreading transformants on plate.

Follow the standard Electroporation protocol, as listed below.

Note that pCP20 has a temperature sensitive origin of replication and requires growth at 30°C for replication to occur. All recovery and plating steps should be done at this temperature.

Protocol

1. If using frozen electrocompetent cells, remove aliquot(s) from the -80°C freezer to thaw on ice for 5 to 10 minutes.
2. To the thawed cell-slurry, add ≈ 1-2 µL of purified DNA (≈50-100 ng of plasmid) and allow mix to sit on ice for 5 minutes.
3. Minimizing mixing, transfer the complete cell slurry to a pre-chilled 1cm gap cuvette. If preparing multiple strains for transformation, keep filled cuvettes on ice until ready to electroporate.
4. Remove cuvette from ice, wipe the metal plates with a dry kim-wipe, and place in the electroporation chamber.
5. Perform the electroporation at 1.8 kV and record the time constant $\tau$. Efficient transformation will occur with time constants 5 ms ≤ $\tau$ ≤ 6ms.
6. Immediately remove the cuvette from the electroporator and add ≈ 1 mL of LB or SOC to the cuvette, mix, and transfer to a glass test tube.
7. Place the culture in a waterbath shaker and allow to recover for ≥ 1 hr. At this time, put an LB agar plate (with appropriate selection, if desired) in an incubator to warm it up.
8. After the recovery period is complete, spread 10µL - 100µL of culture onto plates and allowed to grow for ≥ 14 hours at an appropriate temperature.
9. Store the remainder of the transformed culture at 4°C. If you don’t have colonies on your plate, you can try concentrating and plating the remainder of the culture.

Step 2: Inducing FLP Recombination

Materials & Equipment

Material	Amount	Function
pCP20 Harboring Strain	1 colony	Strain to be recombined
https://www.notion.so/Lysogeny-Broth-LB-897ba670d72745ca934f44dd64c87c42	5 mL per colony	Culture
Glass beads / spreader	—	For spreading recombinants on a plate.

This step triggers recombination to occur by expressing the flp gene under a temperature shock. The trick is that this temperature shock, in addition to triggering recombination, will disable the replication of pCP20 and curing it from the strain.

Protocol

1. Pick a single colony from a plate containing the strain to be recombined that harbors the pCP20 plasmid and inoculate 5mL of LB in a large test tube or erlenmeyer flask.
2. Allow the culture to grow at 42°C overnight (or at least until saturation) to allow for recombinants to proliferate.
3. Once saturated, perform a $1:10^6$ dilution into fresh LB. This should be done by perform 3 serial $1:10^2$ dilution series into 5 mL of LB.
4. Onto a pure LB plate, spread 50µL if the $1:10^6$ diluted LB culture and allow to grow overnight at 30° C to prevent loss of pCP20 from colonies that were not cured.

Step 3: Selecting for Recombinants

Materials & Equipment

Material	Amount	Function
Potentially recombined colonies	1 plate	Colonies to be screened
LB Agar Plate (No Antibiotic)	1 plate (preferably gridded)	Plate to ensure efficient patching
LB Agar Plate with Ampicillin or Carbenicillin	1 plate (preferably gridded)	Plate to ensure loss of pCP20 plasmid
LB Agar plate with selection cassette removed by FLP-FRT process	1 plate (preferably gridded)	Plate to ensure loss of selection cassette that was flanked by FRT sequences
Inoculation loops	10 - 20 (1 per colony)	For selecting and patching colonies across plates.

This step is a manual procedure to screen through colonies produced in step 2. Successful recombinants will only grow on an LB agar plate, and not on an ampicillin plate (indicating that they’ve lost the pCP20 plasmid) nor a plate with the original selection cassette (indicating that the selection marker has been removed from the chromosome). You should perform colony PCR on final candidate recombinants and send the segment for sequencing to ensure you have what you think you have.

Protocol

1. Set up the three agar plates and label regions of the plate where you will patch colonies. Gridded plates are ideal for this as they have ≈25 individual transparently labeled squares on the bottom.

2. Select a potential recombinant colony from the plate from step 2 and pick it up with the end of a sterile inoculation loop.

3. In the following order, draw a small stripe on an annotated section of each plate with the tip of the loop, starting with your (hopefully removed selection marker), followed by the ampicillin plate, and then finally the LB plate (to ensure that you didn’t run out of cells). Do not pick a new colony for each plate.

4. Repeat step 3 for ≈ 10 colonies.

   ⚠️ If using NCM3722, you will need to strike ≈ 30 colonies per recombinant. For some unknown reason, FLP-FRT recombination is inefficient in this strain. ⚠️

5. Place the plates in the incubator at 30°C and allow the plates to grow for ≈ 16 - 24 hours.

6. Once grown, examine each plate. Colonies that were successfully recombined will only grow on the LB plate. Select 1 or 2 candidate colonies, touch the lawn on the LB stripe with a sterile inoculation loop, and swirl in 3 mL of sterile LB growth medium.

7. Allow culture(s) to grow to saturation, and freeze each in 50% glycerol, and enter into the strain databse. Congrats, you’ve got a recombinant 🎉.