Plasmid 25769: pKOV

• Gene/insert name: None
• Alt name: pKOV gene replacement vector
• Alt name: pKO5
• Vector backbone: pMAK700, pMAK705, pBS-TS
  (Search Vector Database)
• Backbone manufacturer: N/A
• Vector type: Bacterial Expression
• Backbone size (bp): 8673
• 5' sequencing primer: pK03-L: 5'-AGGGCAGGGTCGTTAAATAGC-3'
• 3' sequencing primer: pK03-R: 5'-TTAATGCGCCGCTACAGGGCG-3'
• Bacterial resistance(s) Chloramphenicol
• Growth strain(s) DH5alpha
• Growth temperature (℃): 30
• Growth instructions: Grow at 30 degrees Celsius
• High or low copy: Unknown
• Sequence: View sequences (4)
• Supplemental document: Gene replacement using pKO vectors
• Principal Investigator: George Church
• Terms and Licenses: MTA

Comments: 

pKOV is identical to the pKO3 vector described in J. Bacteriology 179: 6228-6237, except for the addition of a 3kb stuffer sequence in the multiple cloning site. This stuffer permits (i) directional cloning using NotI and BamHI and (ii) clean separation of doubly cut vector from singly cut contaminants when using this pair of enzymes. The pKOV cloning site is: 5' - SmaI - NotI - SmaI- stuffer - BamHI - SalI - 3'. BamHI and SalI are not used together, nor is SmaI used with NotI. BglII & BclI cut ends are compatible with BamHI. PmeI & SwaI are compatible with SmaI.

NOTE: pKOV has a temperature sensitive pSC101 replication origin. To recover the plasmid, strains harboring the plasmid must be grown at 30 deg C under chloramphenicol selection.

Gene replacement: Mutant alleles cloned into the pKOV gene replacement vector are electroporated into recombination proficient strains (eg. EMG2) and allowed to recover for 1 h at 30 deg C. The cells are plated on prewarmed chloramphenicol/LB plates and incubated at 42 deg C. To measure the integration frequency, the electroporated cells are also plated on chloramphenicol/LB plates at 30 deg C. From the 42 deg C plate, 1-5 colonies are picked into 1 ml of LB broth, serially diluted, and immediately plated at 30&degree;C on either 5% w/v sucrose or 5% sucrose+antibiotic plates. The 5% sucrose plates are replica plated to chloramphenicol plates at 30 deg C to test for loss of the replacement vector (cms). The gene replacement is confirmed by either PCR using primers flanking the targeted open reading frame or by genomic Southern's.

Article: Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. Link et al (J Bacteriol. 1997 Oct . 179(20):6228-37. PubMed)