E. coli BW25141 (rrnB3 DElacZ4787 DEphoBR580 hsdR514 DE(araBAD)567 DE(rhaBAD)568 galU95 DEendA9::FRT DEuidA3::pir(wt) recA1 rph-1) was used for maintenance of the template plasmid pKD13 (GenBank™ Accession number AY048744). pKD46 (GenBank™ Accession number AY048746; Datsenko and Wanner, 2000 (link)) was made by PCR amplification of the Red recombinase genes from phage λ and cloning into pKD16, a derivative of INT-ts (Haldimann and Wanner, 2001 (link)) carrying araC and araBp from pBAD18 (Guzman et al, 1995 (link)).
Protocol detail
Genetic Manipulation of E. coli
E. coli BW25141 (rrnB3 DElacZ4787 DEphoBR580 hsdR514 DE(araBAD)567 DE(rhaBAD)568 galU95 DEendA9::FRT DEuidA3::pir(wt) recA1 rph-1) was used for maintenance of the template plasmid pKD13 (GenBank™ Accession number AY048744). pKD46 (GenBank™ Accession number AY048746; Datsenko and Wanner, 2000 (link)) was made by PCR amplification of the Red recombinase genes from phage λ and cloning into pKD16, a derivative of INT-ts (Haldimann and Wanner, 2001 (link)) carrying araC and araBp from pBAD18 (Guzman et al, 1995 (link)).
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Corresponding Organization :
Other organizations : Keio University, Nara Institute of Science and Technology, Japan Science and Technology Agency, Purdue University West Lafayette
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Variable analysis
independent variables
- Introduction of the pKD46 plasmid into E. coli BW25141
- Maintenance of the template plasmid pKD13
- E. coli BW25141 strain characteristics: rrnB3 DElacZ4787 DEphoBR580 hsdR514 DE(araBAD)567 DE(rhaBAD)568 galU95 DEendA9::FRT DEuidA3::pir(wt) recA1 rph-1
- Positive control: pKD46 plasmid, made by PCR amplification of the Red recombinase genes from phage λ and cloning into pKD16, a derivative of INT-ts carrying araC and araBp from pBAD18
- Negative control: Not explicitly mentioned
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