The Phage Cookbook by Dominik Refardt


Red swap (λ Red-promoted gene replacement with PCR products)

Background

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.

PCR

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.

Primer Sequence
P1Tobi 5'-gtgtaggctggagctgcttcg-3'
P2Tobi 5'-ctaaggaggatattcatatg-3'
P1pKD3noFRT 5'-AAATGGCGCGCCTTACGCCC-3'
P2pKD3noFRT 5'-CGCGCCTACCTGTGACGGAA-3'
P1pKD4noFRT 5'-TCACGCTGCCGCAAGCACTC-3'
P2pKD4noFRT 5'-AGCGCTTTTGAAGCTGGGGTG-3'

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.

  • Optimize the PCR (temperature, primer concentration, Mg2+ concentration).
  • Use a high fidelity polymerase (Pfu) and 40 cycles to make your insert of choice. Have at least 45 bp of homologous 5' ends to the locus you want to insert at.
  • Pool several PCR reactions (2-4 x 100 µL), purify with your kit of choice and elute with 30 µL water.
  • If you do not use one of the above plasmids as template, but another plasmid, you need to ensure that no plasmids are carried over. Either linearize it before the PCR (and use a low concentration as PCR template) or then digest the PCR product with an appropriate restriction enzyme.

Preparation of competent cells

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).

  1. Grow overnight culture at 30 °C in LB containing 100 µg/mL ampicillin
  2. First thing in the morning, cool down the centrifuge.
  3. Inoculate 50 mL LB containing 100 µg/mL ampicillin and 20 mM arabinose with 500 µL overnight culture (i.e. 1:100 dilution; use flask with baffles). Prior to inoculation, take out 1 mL of LB as a control for the spectrophotometer.
  4. Incubate in shaking water bath or incubator at 30 °C until the OD600 is approximately 0.6 (0.5 – 0.8).
  5. Immediately submerse the flask in ice-water slurry while swirling. Keep swirling for a few minutes. Leave on ice for 10 min.
  6. Transfer culture to a prechilled 50-mL Falcon tube.
  7. Centrifuge 10 min at 4200 g and resuspend pellet in 35 mL ice cold ddH2O.
  8. Centrifuge 10 min at 4200 g and resuspend pellet in 35 mL ice cold ddH2O.
  9. Centrifuge 10 min at 4200 g and resuspend pellet in 2 ml ice cold 10 % glycerol.
  10. Centrifuge 10 min at 4200 g and resuspend pellet in 500 µL ice cold 10% glycerol.
  11. Use cells directly for electroporation (keep on ice!), or freeze aliquots of 90 µL at -80 °C. Best to quick chill prior to freezing with liquid nitrogen (or have 1.5-mL tubes ready that are chilled to -80 °C).

Transformation and selection

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.

  1. If competent cells are frozen, thaw them on ice. Unpack the electroporation cuvettes and chill them on ice. Label the 2-mL tubes.
  2. Add an aliquot of purified PCR product (1-6 µL) to 90 µL of competent cells.
  3. Transfer the competent cells into an electroporation cuvette.
  4. Electroporate with 1.80 mV setting.
  5. Recover cells immediately in 1 mL warmed SOC or LB, and incubate shaking for 1 h at 37 °C (now you don't need the pKD46 plasmid anymore).
  6. Plate 100 µL of culture on LB plates containing the appropriate antibiotic (50 µg/mL kanamycin or 10 µg/mL chloramphenicol)
  7. Centrifuge the remaining 900 µL (5 min, 8000 g), remove 800 µL supernatant, resuspend pellet in remaining 100 µL, and plate on selective plates (you can also skip the centrifugation step, plate all 900 µL, and dry the plate close to a flame).
  8. Incubate over night at 37 °C (or longer).
  9. Pick resistant colonies and restreak on new selective plates.
  10. Incubate over night at 37 °C
  11. Pick colonies, streak on new selective plates, and confirm genotype by PCR (one primer in the insert, one outside of it).
  12. Incubate over night at 37°C
  13. Pick a single colony and inoculate in 3 mL LB with the appropriate antibiotic.
  14. Incubate over night at 37 °C
  15. Make 15% glycerol stock (300 µL 50% glycerol + 700 µL culture) and store at -80 °C.
  16. Confirm the loss of pKD46 by plating 100 µL of the over night culture on Ampicillin plates, incubate over night at 37 °C. Not a single colony should grow.
It may well happen that it doesn't work the first time you try. If this is the case, try again. Optimize as much as you can, prepare highly concentrated electro-competent cells, produce a lot of PCR-product, and talk to your cells in soothing voice.

Move construct to fresh background

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.

Deletion of resistance markers using pCP20/flp-recombinase

Requires plasmid pCP20, which contains a temperature-inducible flp gene (Cherepanov & Wackernagel 1995). The plasmid also confers antibiotic resistance (ampicillin, chloramphenicol).

  1. Prepare competent cells of your obtained clone(s) (harboring the resistance cassette) as described above, except growing the culture at 37 °C.
  2. Add aliquot of purified pCP20 to competent cells, mix.
  3. Electroporate and recover as described above.
  4. Plate 100 µL of culture on LB plates containing 50 µg/mL ampicillin.
  5. Centrifuge the remaining 900 µL (5 min, 8000 g), remove 800 µL supernatant, resuspend pellet in remaining 100 µL, and plate on selective plates (you can also skip the centrifugation step, plate all 900 µL, and dry the plate close to a flame).
  6. Incubate over night at 30 °C.
  7. Pick approximately 20 colonies and streak them on LB plates without antibiotic.
  8. Incubate over night at 37 or 43 °C to activate Flp-recombinase.
  9. Select those clones that lost all antibiotic resistance (the majority should loose the resistance cassette and the plasmid pCP20), purify once on LB, do colony PCR to confirm genotype (using suitable deletion-specific primers) and purify PCR-positive clones on LB plates (without antibiotics).
  10. Incubate over night at 37 °C, confirm loss of resistances again (ampicillin ofr pCP20, kanamycin or chloramphenicol for the resistance cassette). Make Stock.