Keio and ASKA collections

The Keio collection consists of single-gene knockout strains in the K12 BW25113 strain background (an MG1655 derivative). They contain a Kan^R resistance cassette in place of the gene. One can easily remove this cassette to create an in-frame deletion of the gene using FLP recombinase expression from plasmid pCP20.

The ASKA collection is a set of ORFs cloned from E. coli K12 W3110 into the high copy number plasmid pCA24N. This plasmid has a modified pMB1 replication origin (same as pQE30 from Qiagen) and Cam^R marker. The cloned ORF is under control of the IPTG-inducible T5-lac promoter.

Our samples of these strain collections were transferred from 96-well microplates into 384-well microplates using a 96-well pin tool. Thus, the plates are interspersed with one another, with one plate in the upper-left register, the next plate in the upper right register, the third plate in the lower left register, and the final plate in the lower right register. This is the layout in the order listed on the plate label. The attached spreadsheets can be used to determine if a strain exists in the collection (be sure you search gene synonyms) and to find the well number for a desired strain to retrieve it.

*Important!* When retrieving a strain from the freezer and regrowing it:

1. Streak out to a single colony on an LB agar plate with the correct antibiotic.
2. Verify that the strain/plasmid is correct.
   • By comparing PCR product size or a known phenotype to BW25113 for Keio Strains
   • By sequencing the plasmid insert for ASKA strains. Sequencing primers SPL003 and SPL004 flank the ORF insertion site in the pCA24N.
3. Create a stock of this verified isolate in a cryovial for further use by the lab.

Some known issues with our copy of the Keio collection

• JW0578 ( entF) has some slight contamination from JW3684 ( trmE)
• We were unable to revive JW5830 mntP ( yebN)
• We were unable to revive JW5807 leuB

Keio Revival

1. Spin plate 5 minutes >1k RPM using swinging bucket rotor
2. Pierce the foil lid using P200 tip to touch frozen culture
   • It can be useful to use a sharpie to mark the desired well location
3. Inoculate liquid culture or streak directly in/on media containing Kan
   • If initially inoculating liquid culture, see note above about importance of going through a single colony and verifying via PCR or phenotype for expected strain
4. Carefully roll existing lid back upon itself with force directed straight down to avoid splashing and add new foil lid WHICH CAN HANDLE −80°C when plate is free of condensation
   • Alternatively, if cover is in decent shape (defined as a few punctures in it, and individual wells easily discernible through a small number of foil lids), a fresh additional lid can be added directly atop the existing lid once condensation has stopped collecting on plate.
5. Be sure to use a lid roller to get good seal!