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