Recombineering Website

Welcome to the official, non-profit, website for recombineering!

• What's Recombineering?
• To Start Recombineering
• List of Available Reagents
  • Bacterial strains
  • Plasmids

On this website you will find a number of tested and detailed protocols for doing recombineering, as well as plasmid maps, plasmid sequences, references to recombineering publications, and information on how to obtain the recombineering reagents from us.

What's Recombineering?

Recombineering (recombination-mediated genetic engineering) is a powerful method for fast and efficient construction of vectors for subsequent manipulation of the mouse genome or for use in cell culture experiments. It is also an efficient way of manipulating the bacterial genome directly.

Recombineering is a method based on homologous recombination in E. Coli using recombination proteins provided from λ phage.

Our bacterial strains contain a defective λ prophage inserted into the bacterial genome. The phage genes of interest, exo, bet, and gam, are transcribed from the λPL promoter. This promoter is repressed by the temperature-sensitive repressor cI857 at 32°C and derepressed (the repressor is inactive) at 42°C. When bacteria containing this prophage are kept at 32°C no recombination proteins are produced. However, after a brief (15 minutes) heat-shock at 42°C a sufficient amount of recombination proteins are produced. exo is a 5'-3' exonuclease that creates single-stranded overhangs on introduced linear DNA. bet protects these overhangs and assists in the subsequent recombination process. gam prevents degradation of linear DNA by inhibiting E. Coli RecBCD protein.

Linear DNA (PCR product, oligo, etc.) with sufficient homology in the 5' and 3' ends to a target DNA molecule already present in the bacteria (plasmid, BAC, or the bacterial genome itself) can be introduced into heat-shocked and electrocompetent bacteria using electroporation. The introduced DNA will now be modified by exo and bet and undergo homologous recombination with the target molecule. The method is so efficient that co-electroporation of a supercoiled plasmid and a linear piece of DNA into heat-shocked, electrocompetent bacteria will work as well.

To Start Recombineering

You can find a number of tested and detailed protocols for doing recombineering on this website. You can also find information about plasmid maps, plasmid sequences, references to recombineering publications, and how to obtain the recombineering reagents from us.

List of Available Reagents

If you're interested, and are at a not-for-profit institution, we will ship you the reagents you need, please visit How to order reagent section for application procedure.

Below is a list of all strains and plasmids described in publications from this laboratory.

Please note that the strains DY380, EL250, and EL350 are no longer available. They were replaced by the new strains SW102, SW105, and SW106, respectively. The new strains are identical to the old ones, except they have been modified to also allow for efficient BAC modification using our new galK positive/negative selection procedure.

• Bacterial strains

  1. DY380 (not available) (ref): The original, DH10B-derived strain. These bacteria contain a defective λ prophage with recombination proteins exo, bet, and gam being controlled by the temperature-sensitive repressor cI857. The bacteria are tet resistant (12.5 μg/ml). Genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZ M15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu) 7649 galU galK rspL nupG [ λcI857 (cro-bioA) <> tet]
  2. EL250 (not available) (ref): Derived from DY380. In addition to the recombination genes found in DY380 EL250 also contains a tightly controlled arabinose-inducible flpe gene. flpe will mediate recombination between two identical frt sites. This strain is not tet resistant. Genotype: DY380 [(cro-bioA) <> araC-PBADflpe]
  3. EL350 (not available) (ref): Derived from DY380. In addition to the recombination genes found in DY380 EL350 also contains a tightly controlled arabinose-inducible cre gene. cre will mediate recombination between two identical loxP sites. This strain is not tet resistant. Genotype: DY380 [(cro-bioA) <> araC-PBADcre]
  4. SW102 (ref): Derived from DY380 and has all the same features, but designed for BAC recombineering using galK positive/negative selection. These bacteria contain a fully functional gal operon, except the galK gene is missing. The ability to grow on galactose minimal medium can be restored by adding galK in trans, by inserting a galK expression cassette into a BAC. Next, the galK cassette can be replaced by any dsDNA carrying any desired mutation, by selection against galK using 2-deoxy-galactose.
  5. SW105 (ref): Derived from EL250. Like SW102, these cells can be used for galK positive/negative selection. Like EL250, these cells contain an ara-inducible Flpe gene.
  6. SW106 (ref): Derived from EL350. Like SW102, these cells can be used for galK positive/negative selection. Like EL350, these cells contain an ara-inducible Cre gene.

• Plasmids (for maps and sequences, please follow the link).

  1. PL451: A plasmid containing a neo cassette flanked by two frt sites and one loxP site. The neo gene is expressed both from a prokaryotic promoter (em7) and a eucaryotic promoter (Pgk): Frt-Pgk-em7-Neo-Frt-loxP. (ref)
  2. PL452: A plasmid containing a neo cassette flanked by two loxP sites. The neo gene is expressed both from a prokaryotic promoter (em7) and a eucaryotic promoter (Pgk): loxP-Pgk-em7-Neo-loxP. (ref)
  3. PL253: A pBluescript-derived plasmid for retrieval of DNA from a BAC. This plasmid contains an Mc1-driven Thymidine Kinase (TK) cassette for negative selection in ES cells. Retrieval of DNA into this vector from a BAC is the first step in the construction of a gene targeting vector. (ref)
  4. pIGCN21: A plasmid containing the IRES-EGFPcre-Frt-kan-Frt cassette. This can be used to insert an EGFP-cre fusion gene into a BAC under the control of a gene of interest to make a tissue-specific Cre mouse. (ref)
  5. pEL04: A plasmid containing the Cam-SacB cassette to be used for positive/negative selection. Positive selection using chloamphenicol, and negative selection against the SacB gene using sucrose. (ref)
  6. pTamp: A plasmid containing an amp cassette for replacing the loxP site in pBeloBAC11. (ref)
  7. pGalK: A galK expression vector for use together with either SW102, SW105, or SW106 in galK positive/negative selection-based BAC recombineering. galK is expressed from the procaryotic em7 promoter. Homology arms can be added using PCR or cloned into this cassette using the unique flanking restriction sites. ( ref )