Efficient recombination between short, linear DNA fragments and the bacterial chromosome can be catalyzed by taking advantage of phage recombination systems. The phage λ Red locus encodes a system that promotes homologous recombination. The λ Red locus includes three genes: bet, exo, and gam. Exo is a 5'-3' exonuclease that degrades the 5' ends of linear DNA molecules. Bet is a single-stranded DNA binding protein that binds to the single stranded 3' ends generated by Exo and promotes annealing to complementary DNA. Gam binds to the host RecBCD complex and inhibits its exonuclease activity.
Two independent methods, which take advantage of λ Red-promoted recombination, have been recently developed to promote gene disruptions with PCR products containing very short regions of homology (30 to 50 bp). An exchange made by any of these methods is generically described as a "Red-swap". The method developed by Court and colleagues (Yu et al. 2000) uses a defective prophage, which expresses the bet, gam, and exo genes from λPL under the control of the cI857 (temperature sensitive) repressor. The problem with this system for our purpose is that it includes a defective prophage, which can interfere with what we are doing, as we use phages as well. An alternative method developed by Wanner and colleagues (Datsenko & Wanner 2000), expresses the λ Red genes under the control of an arabinose-inducible promoter from a temperature-sensitive, low copy number plasmid. Expression of the Red functions from the PBAD promoter facilitates tight regulation with induction by the addition of arabinose. This method is described below.
To amplify resistance cassettes, use plasmids pKD3 (cat, chloramphenicol resistance cassette) or pKD4 (neo, kanamycin resistance cassette). These plasmids require the pir gene product for replication, which means that a carry-over of the plasmids (and false positives) is not possible. The resistance cassettes on these plasmids are also flanked by FRT sites, which allow the removal of the cassettes once inserted in the bacterial chromosome with a FLP helper plasmid.
To amplify the resistance cassettes, use primers P1Tobi and P2Tobi, which work with both plasmids. Note that P1Tobi is a modification of P1 as given by Datsenko & Wanner (2000), and P2Tobi is downstream of P2. Extend these primers at the 5' end with 45 bp of homology extensions. Alternatively, if you don't want to introduce the FRT-sites, you need to use primers inside these flanking regions. Because plasmids pKD3 and pKD4 differ there, primers are different for the two plasmids.
When designing the homology extensions, try to interfere as little as possible with the E. coli chromosome. Do not disrupt genes such that they are still expressed but not complete, leave terminators intact so that transcription ends properly, do not interfere with promoters that might be needed by the adjacent gene, etc. If you need more information on E. coli genes, use EcoCyc and PEC, to check for regulatory elements, use RegulonDB (mirror). And keep in mind that the homology extensions will also show up in your construct (i.e. if you want to knockout a gene, the extension must be outside of it, not at its end).
Finally, about the correct insertion of the resistance cassette: In pKD4, it is P1 -> kanamycin-resistance cassette -> P2. In pKD3, it is the other way round, namely P2 -> chloramphenicol-resistance cassette -> P1. In many cases this is probably not important, but sometimes you may care about the direction with which the cassette is inserted.
Plasmids pKD46, pKD78, and pKD119 carry the λ red genes behind the araBAD promoter. Expression of the λ red genes is sufficiently induced by adding 0.1% of L-arabinose to your growing culture. The plasmids themselves are temperature-sensitive to be easily cured from your strain. By transforming any E.coli K-12 strain with one of those three plasmids, it turns them into highly recombination efficient strains when adding arabinose. But you have to grow them at max. 32 °C to maintain the plasmid! pKD46 carries the bla gene (ampicillin resistance), pKD78 carries the cat gene (chloramphenicol resistance), pKD119 carries the tet gene (tetracycline resistance).
You need selective plates with the respective antibiotic, autoclaved ddH2O and autoclaved 10% glycerol. You also need chilled cuvettes, pipettes (25 and 10-mL), 50-mL Falcon tubes, 14-mL Falcon tubes, and 1.5 mL tubes (sterile).
You need chilled electroporation cuvettes (1 cm), chilled 1-mL (blue) and 200-µL (yellow) pipette tips, warm (37 °C) LB or SOC and 2-mL tubes. When transferring cells, use a blue pipette tip and make the bore wider by cutting off the front.
After successful electroporation and recombination, the construct has to be moved to a fresh background (e.g. MG1655 or any desired E.coli K12 strain) by P1 transduction because λ red-mediated recombination can introduce further changes in the genome.
Requires plasmid pCP20, which contains a temperature-inducible flp gene (Cherepanov & Wackernagel 1995). The plasmid also confers antibiotic resistance (ampicillin, chloramphenicol).
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