FLP Recombination in E. coli

This procedure is commonly used to eliminate the Kanamycin resistance cassette from E. coli strains from the Keio collection or produced by P1 transduction from Keio strains or the i>Deconvoluter library.

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.
   • Best to avoid placing plates in top shelf of stacked small shaking incubator as temperature may be slightly higher than expected.

Expected results:

1. 50-100 colonies expected by 24 hours.
2. Plate may take 18+ hours to be able to see colonies. DO NOT assume failure if no colonies seen first thing in morning. Cells grow more slowly at 30°C.

Day 2: Allow recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

NOTE: To save a day, you can inoculate several colonies into 5mL LB at the same time that you patch them (do the procedure for Day 5 on Day 4). You will proceed with the liquid LB cultures that correspond to colonies that were Kan and Amp sensitive. If you do this, be sure to only use colonies that have growth on the LB-only plate, in addition to no growth on the LB-Kan and LB-Amp plates.

Expected results: Most if not all patches should be completely Kanamycin and Ampicillin sensitive.

Day 5: Grow up successful recombinants (can be combined with Day 4, see note above)

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 0008.
3. Cherepanov, P. P., Wackernagel, W. (1995) Gene disruption in Escherichia coli: Tc^R and Km^R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158: 9–14.
4. Keio Strain construction details

FLP Recombination in E. coli

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.
   • Best to avoid placing plates in top shelf of stacked small shaking incubator as temperature may be slightly higher than expected.

Expected results:

1. 50-100 colonies expected by 24 hours.
2. Plate may take 18+ hours to be able to see colonies. DO NOT assume failure if no colonies seen first thing in morning. Cells grow more slowly at 30°C.

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

Day 5: Grow up successful recombinants (can be combined with Day 4, see note above)

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 0008.
3. Cherepanov, P. P., Wackernagel, W. (1995) Gene disruption in Escherichia coli: Tc^R and Km^R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158: 9–14.
4. Keio Strain construction details

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.
   • Best to avoid placing plates in top shelf of stacked small shaking incubator as temperature may be slightly higher than expected.

Expected results:

1. 50-100 colonies expected by 24 hours.
2. Plate may take 18+ hours to be able to see colonies. DO NOT assume failure if no colonies seen first thing in morning. Cells grow more slowly at 30°C.

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

• NOTE: To save a day, you can inoculate several colonies into 5mL LB at the same time that you patch them (do the procedure for Day 5 on Day 4). You will proceed with the liquid LB cultures that correspond to colonies that were Kan and Amp sensitive. If you do this, be sure to only use colonies that have growth on the LB-only plate, in addition to no growth on the LB-Kan and LB-Amp plates.

Expected results: Most if not all patches should be completely Kanamycin and Ampicillin sensitive.

Day 5: Grow up successful recombinants (can be combined with Day 4, see note above)

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.

1. Keio Strain construction details

FLP Recombination in _E. coli

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.

1. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.

1. Plate may take 18+ hours to be able to see colonies. DO NOT assume failure if no colonies seen first thing in morning. Cells grow more slowly at 30°C.

Day 2: Induce recombination

1. Inoculate into 5 ml of LB in a test tube.
2. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

Day 5: Grow up successful recombinants (can be combined with Day 4, see note above)

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

FLP Recombination in _E. coli

Commonly used to eliminate the Kanamycin resistance cassette from E. coli strains from the Keio collection or produced by P1 transduction of the i>Deconvoluter library.

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Expected results:

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

FLP Recombination in _E. coli

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Expected results:

1. 50-100 colonies expected by 18-20 hours.
2. Plate may take 18+ hours to be able to see colonies. DO NOT assume failure if no colonies seen first thing in morning

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

• NOTE: If 5mL LB is inoculated for each colony, a day can be saved. If LB culture grows, but LB plate does not, left with a choice of if you believe patching was bad or contamination.

Expected results: Most if not all patches should be completely Kanamycin and Ampicillin sensitive.

Day 5: Grow up successful recombinants (can be skipped, see note in Day 4)

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

FLP Recombination in _E. coli

Commonly used to eliminate the Kanamycin resistance cassette from E. coli strains from the Keio collection.

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Expected results:

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

• NOTE: If 5mL LB is inoculated for each colony, a day can be saved. If LB culture grows, but LB plate does not, left with a choice of if you believe patching was bad or contamination.

Expected results: Most if not all patches should be completely Kanamycin and Ampicillin sensitive.

Day 5: Grow up successful recombinants (can be skipped, see note in Day 4)

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

FLP Recombination in _E. coli

Commonly used to eliminate the Kanamycin resistance cassette from E. coli strains from the Keio collection.

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and incubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Expected results:

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

FLP Recombination in _E. coli

Commonly used to eliminate the Kanamycin resistance cassette from E. coli strains from the Keio collection.

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.

1. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
2. Grow overnight at 30°C.

Expected results:

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates. Do the patching by picking a colony and then streaking a small spot on each plate in this order. Be sure you patch on LB last. Failure to grow can sometimes occur because no cells were transferred to the later plates. This order ensures that, if you get the desired growth on the last plate and no growth on the LB + Kan and LB + Amp plates, it was not due to "running out" of cells in the later patches.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

Expected results: Most if not all patches should be completely Kanamycin and Ampicillin sensitive.

Day 5: Grow up successful recombinants

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

FLP Recombination in _E. coli

Commonly used to eliminate the Kanamycin resistance cassette from E. coli strains from the Keio collection.

Day 1: Transform recombinase plasmid

1. Make electrocompetent cells of the desired E. coli strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and inclubate in a 1.7 ml tube shaking on its side at ≥120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Day 2: Induce recombination

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB in a test tube.
3. Grow overnight at 43°C to induce FLP recombinase expression and select for loss of pCP20.

Day 3: Plate to get single candidate recombinants

1. Make a 10⁶ dilution of the overnight culture via 3 wet DTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of this dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Expected results: ~200 colonies on LB plates.

Day 4: Screen for genomic recombination and plasmid loss

1. Grow overnight at 37°C for LB and LB + Kan and grow at 30°C for LB + Amp plates.

Day 5: Grow up successful recombinants

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6: Archive clones

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

1. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.

1. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
2. Grow overnight at 30°C.

1. Pick a single colony from the LB + Amp plate.

1. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

Eliminating the Kanamycin Resistance Cassette from Keio Strains

Day 1

1. Make electrocompetent cells of the desired strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and inclubate in a 1.7 ml tube shaking on its side at 120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Day 2

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB.
3. Grow overnight at 43°C to select for loss of pCP20.

Day 3

1. Make a 10⁶ dilution of the overnight culture via 3 wet dTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Day 4

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 43°C for LB + Amp plates.

Day 5

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

Eliminating the Kanamycin Resistance Cassette from Keio Strains

Day 1

1. Make electrocompetent cells of the desired strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and inclubate in a 1.7 ml tube shaking on its side at 120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Day 2

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB.
3. Grow overnight at 43°C to select for loss of pCP20.

Day 3

1. Make a 10⁶ dilution of the overnight culture via 3 wet dTs (100 µl into 9.9 ml each time).
2. Plate 50 µl of dilution on LB. This should yield a couple hundred colonies.
3. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Day 4

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates.
2. Grow overnight at 37°C for LB and LB + Kan and grow at 43°C for LB + Amp plates.

Day 5

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

Eliminating the Kanamycin Resistance Cassette from Keio Strains

Day 1

1. Make electrocompetent cells of the desired strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and inclubate in a 1.7 ml tube shaking on its side at 120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Day 2

1. Pick a single colony from the LB + Amp plate.
2. Inoculate into 5 ml of LB.
3. Grow overnight at 43°C to select for loss of pCP20.

Day 3

1. Grow overnight at 30°C to prevent partial loss of plasmid from colonies founded by cells that did not lose plasmid.

Day 4

1. Patch six individual colonies from this plate onto LB + Kan, LB + Amp, and LB plates.

Day 5

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

Day 6

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

Eliminating the Kanamycin Resistance Cassette from Keio Strains

Day 1

1. Make electrocompetent cells of the desired strain.
2. Transform with pCP20. This plasmid has a temperature-sensitive origin of replication, confers ampicillin resistance, and encodes the FLP recombinase.
3. Add 500 µl SOC and inclubate in a 1.7 ml tube shaking on its side at 120 rpm for 1 hr at 30°C to allow antibiotic resistance induction.
4. Plate 20 µl of the culture on an LB + Amp plate, save the rest for additional plating if necessary.
5. Grow overnight at 30°C.

Day 2

1. Pick a single colony from the LB + Amp plate.

Day 3

Day 4

1. Inoculate 5 ml LB from patches on LB plates that score as sensitive to both antibiotics.
2. Grow overnight at 37°C, shaking at 120 rpm.

1. Freeze and archive copies of the newly created strain, there the Kan cassette has been eliminated from between the FRT sites. To guard against mutations that may occur during strain construction, it may be wise to archive and test the fitness of several separate colonies (patches).

Sources

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details

1. Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645.
2. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 0008.
3. Keio Strain construction details