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- Construct integration cassette (no known hard size limit, but probably < 5 kb is most efficient). It needs to consist of:
- 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.
- 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).
- 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.
- Other genes/DNA of interest.
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- 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.
- 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).
- 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).
- 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).
- Pick a single colony; grow overnight at 37°C.
- Confirm loss (curing) of pKD46 plasmid by plating some on an amp plate and letting grow overnight at 37°C.
- 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.
- 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.
- Pick 4–16 colonies, grow it up at 37°C with NO antibiotics.
- 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)
- Plate some of the culture at 42°C with no antibiotics.
- Test individual colonies for loss of all antibiotics via plating tests.