Description

CGSC Strain#: 7636
Strain Designation: BW25113      Source of Strain: B.L. Wanner
Other Designations: ME9062
Sex: F- Chromosomal Markers: Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ^-, rph-1, Δ(rhaD-rhaB)568, hsdR514
Strain Comments:

• This is the parent strain for the Keio Collection of single gene knockouts

• This strain was originally reported to be lacI^Q, but it was later shown to be lacI⁺ (Baba 2006 ref. Supplementary Table 1)

• Δ(araD-araB)567-- This deletion extends from ~25 bp upstream of the araB start codon to ~8 bp into the beginning of the araD gene

• ΔlacZ4787(::rrnB-3)-- : 4 tandem copies of the rrnB transcriptional terminator inserted by gene replacement into the region extending from near the SacII site near the N-terminus of lacZ through the promoter.

• rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

• Δ(araD-araB)567 was formerly called Δ araBAD_AH33

• Δ(rhaD-rhaB)568 was formerly called Δ rhaBAD_LD78

• ΔlacZ4787(::rrnB-3) was formerly called Δ lacZ_WJ16

• ΔlacZ4787(::rrnB-3) was formerly called ::rrnB-4

• ::rrnB-3 = 4 copies of rrnB inserted

• Datsenko, KA, BL Wanner 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97(12):6640-5.

• Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1-11

Recovery

1.Obtainan LB agar plate with the appropriate antibiotic.

2.Usinga sterile pipette tip, touch the bacteria growing within the punctured area ofthe stab culture. (A sterilized wire loop or sterile toothpick can be used inplace of a sterile pipette tip.)

3.Runthis tip lightly over a section of the plate, as shown in the figure, to createstreak #1.

4.Usinganother sterile pipette tip, pass through streak #1 and spread the bacteriaover a second section of the plate, to create streak #2.

5.Usinga third sterile pipette tip, pass through streak #2 and spread the bacteriaover the last section of the plate, to create streak #3.

6.Growovernight in a 37 P^oP Cincubator (unless a different growth temperature is indicated on the plasmiddatasheet).

7.Inthe morning, single colonies should be visible. If the bacterial growth is toodense, re-streak onto a new agar plate to obtain single colonies.