Description

The E. coli K-12 BW25113 parent strain is the common background genotype used for the generation of the E. coli Keio Knockout Collection, which is a set of precisely defined,single-gene deletions of all nonessential genes in E.coli K-12. Out of 4288 genes targeted, mutants were obtained for 3985 genes. Two independent mutantswere obtained for each deleted gene, yielding a total of 7970 knockout strains(Baba et al, 2006).

Using λ Red recombination, a FRT-flanked kanamycin cassette wasutilized to replace each coding region. This cassette may be excised by FLPrecombination, leaving an in-frame, translatable sequence that includes theendogenous start, a short recombinational scar sequence, and an 18-nucleotide,C-terminal coding sequence from the endogenous gene. For details concerningcassette excision, please consult (Datsenko and Wanner, 2000).

This collection is anew resource for systematic analyses of unknown gene functions and generegulatory networks, but also for genome-wide testing of mutational effects ina common strain background.

Genotype of BW25113:

rrnB3 ΔlacZ4787 hsdR514Δ(araBAD)567 Δ(rhaBAD)568 rph-1.

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.