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  1. Construct integration cassette (no known hard size limit, but probably < 5 kb is most efficient). It needs to consist of:
    1. 5’ and 3’ regions that overlap with genomic sequence flanking the genomic position of insertion. For example, you could use the 5′ UTR and 3′ UTR of uidA, if you planned on deleting/replacing uidA CDS. According to the original paper, these overlapping regions need only be 40–50 bp long, allowing addition to amplified DNA via oligos <60 bp.
      (question) For cassettes longer than around 3 kb, much longer homology region around 500 bp are needed since it must use a different recombination mechanism.
    2. A selection/screening marker, commonly an antibiotic resistance cassette (that functions at a known, typically lower, cantibiotic at genomic copy#). A fluorescent protein (gfp) or dye-producing enzyme (lacZα or uidA) cassette can be included or even tagged to an antibiotic resistance CDS to allow secondary visual confirmation of integration. Ideally, you have FRT (Flp recombinase target) sites around the marker to allow later removal of the marker with the site-specific Flp recombinase, without which a secondary λred integration with a different resistance gene or CRISPR/Cas is needed to remove the resistance marker. FRT-flanked ChlR and KanR markers can be amplified (even using primers with genomic homologies) from the R6Kγ conditional origin plasmids pKD3 and pKD4, respectively.
    3. Optionally, other genes/DNA of interest.

      [5′ genomic homology] – [Opt. other DNA] – [FRT] – [selection/screening marker(s)] – [FRT] – [Opt. other DNA] – [3′ genomic homology]
  2. Transform strain of interest with pKD46[AmpR], which has the phage λ recombination gene operon, λ red, encoding Exo, Beta, and Gam, controlled by an arabinose-inducible promoter and on temperature-sensitive pSC101 replicon (AmpR for pKD46). Grow plates at 30°C to maintain the temp-sensitive plasmid.
  3. Make a (small scale - see below) electrocompetent cell prep of the pKD46 transformed cells: grow a seed culture at 30°C in 2×YT to an OD≈1, then induce with 1–50 mM (0.05–0.7%) arabinose (Full induction is 0.32%, 16 µL/mL of 20% arabinose, though less, say 1 mM/0.05%, also works) and grow for 1 hr. (The 2×YT and high OD are important - 3 hrs growth if you start off the culture with a 1:30 dilution of overnight seems to work well).
  4. Transform pKD46 competent strain with linear dsDNA (as from PCR), allow to recover for an hour at 37°C, plate on antibiotic used in integration cassette, and grow at 37°C (or 42°C).
  5. Confirm presence of desired sequence by appropriate colony PCRs and presence of desired sequence at targeted locus. (Choose primers across integration boundaries.)
  6. Grow a few colony-PCR-verified isolate in liquid culture to saturation at 37°C to cure away the λred plasmid.
  7. Confirm loss (curing) of pKD46 plasmid by inoculating amp solid or liquid medium with 
  8. At this point, phage P1 transduction can optionally be used to transfer the desired genotype to a clean genetic background (as genomic rearrangements may have been induced by the λred).
  9. Make a competent prep with pKD46-cured cells, then transform with pCP20 which has FRTs, is AmpR, ChlR, and temperature sensitive. For best Plate on LB amp, and grow up at 30°C.
  10. Pick 4–16 colonies, grow it up at 37°C with NO antibiotics.
    1. DO NOT SKIP THIS STEP: Always grow up in media at 37°C first. Otherwise you will not enrich for the right pCP20 clones. (Confirmed by Josh)
  11. Plate some of the culture at 42°C with no antibiotics.
  12. Test individual colonies for loss of all antibiotics via plating tests.
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