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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 nearby genomic sequences (flanking a region to be removed/replaced). For example, you could use the 5’ UTR and 3’ UTR of envZ, if you planned on deleting/replacing envZ. According to the original paper, these overlapping regions need only be some 40–50 bp long. JCA suggests that using the 50 bp overlap method is highly effective.
    2. For cassettes longer than around 3 kb, much longer homology region around 500 bp since it must use a different recombination mechanism (vaguely recall RecA for longer instead of an exonuclease type mechanism).
    3. An antibiotic resistance cassette (that functions at single copy). Ideally, you have FRT sites around the marker for eventual removal of the marker with the site-specific FLP recombinase.
    4. Other genes/DNA of interest.
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  2. Transform strain of interest with pKD46, which has the λ phage recombination genes (λred) on controlled by an arabinose-inducible promoter and a temperature-sensitive replicon (AmpR for pKD46). Grow plates at 30°C to maintain the temp-sensitive replicon.
  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) 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. (Choose primers that convince you of the presence of important pieces, as well as total size).
  6. Pick a single colony; grow overnight at 37°C.
  7. Confirm loss (curing) of pKD46 plasmid by plating some on an amp plate and letting grow overnight at 37°C.
  8. At this point, 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 pKD46). See technique below.
  9. Make a competent prep with pKD46-less cells, then transform with pCP20 (see PMID 7789817), which has FRTs, is AmpR, CamR, and temperature sensitive. 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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