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Recombineering in Escherichia coli is mediated by bacteriophage proteins, either RecE/RecT from Rac prophage [1] or Redαβδ from bacteriophage lambdaλ.[2][3][5] The λ red recombination system is now most commonly used and the first demonstrations of λ red in vivo genetic engineering were independently made by Kenan Murphy[4] and Francis Stewart.[1][2] However, Murphy's experiments required expression of RecA and also employed long homology arms. Consequently the implications for a new DNA engineering technology were not obvious. The Stewart Lab showed that these homologous recombination systems mediate efficient recombination of linear DNA molecules flanked by homology sequences as short as 30 bp into target DNA sequences in the absence of RecA. Now the homology could be provided by oligonucleotides made to order, and standard recA⁻ cloning hosts could be used, greatly expanding the utility of recombineering. Datsenko & Wanner's λ Red recombineering method[5] became the most popular, used in the Keio gene deletion collection, and is the basis of the protocol here: One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products.
To use the λ red recombineering system to modify target DNA (orange), linear donor dsDNA (blue) (ssDNA usage is not covered here) that includes 5′ and 3′ "homology arms" (H1/H2) matching the target DNA is electroporated into E. coli expressing the λ red enzymes, usually encoded on a temporary plasmid. These enzymes then catalyze the homologous recombination of the donor DNA with the target DNA sequence. The homology arms (H1/H2) of the donor DNA must match the region immediately 5′ and 3′ to the target sequence to be inserted into. Sequence in between the homology arms of the donor DNA replaces the sequence between the matching homology sequences in the target DNA. A selection, counterselection, or screening gene marker is usually included to select or screen for recombinants, and these markers are often designed to be removed by a second round of recombineering coupled with counterselection of a counterselectable marker, or using site-specific recombinases like the Flp-FRT system which requires FRT sequences around the region to be removed by Flp recombinase expressed (green) from a second plasmid.
The example below shows how the E. coli "Keio" gene deletion collection was constructed, wherein each non-essential gene in E. coli was deleted while minimizing disruptions to any translationally-coupled genes inside operons by using homology arms that preserve the start codon of the gene B to be deleted and the Shine-Dalgarno sequence of the downstream gene C that is internal to gene B toward its end.
dsDNA Procedure
- Construct integration cassette (no known hard size limit, but probably <5 kb is most efficient). It needs to consist of:
- 5’ and 3’ regions Homology arms: 5′ and 3′ sequences that overlap with genomic target locus sequence flanking the genomic position of insertion. For example, you could use the 5′ UTR and 3′ UTR of uidA lacI, if you planned on deleting/replacing uidA the lacI CDS. According to the original paper, these overlapping regions need only be 40–50 bp long, allowing addition to amplified DNA cassette via oligos <60 primers ≤60 bp.
True True or not? For cassettes larger than around 3 kb, much longer homology region around 500 bp are needed since it must use a different recombination mechanism.
- A selectionSelection/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. If not removed, that resistance cannot be used for subsequent recombineering or plasmid maintenance, limiting strain use. Multiple different FRT-flanked markers may be integrated followed by a single Flp treatment to remove all markers. FRT-flanked ChlR and KanR markers can be amplified (even using primers with genomic homologies) from the R6Kγ conditional origin plasmids pKD3 and pKD4/pKD14, respectively, often successful with primers that include the 40–50 bp genomic homology. Primers must not bind the FRT sites directly as they are repeated and ≈palindromic, and the pKD plasmids have standard priming sites. pKD4 (and not pKD14) leaves a RBS-start codon scar to leave a less disruptive deletion scar inside a polycistronic operon, aiming to preserve downstream CDS expression.
- Insertional sequence: Optionally, other genes/DNA of interest, which remain at the locus after excision of the region between FRTs.
[5′ genomic homology] – [opt. other DNA] – [FRT] – [selection/screening marker(s)] – [FRT] – [Opt. other DNA] – [3′ genomic homology]
- 5’ and 3’ regions Homology arms: 5′ and 3′ sequences that overlap with genomic target locus sequence flanking the genomic position of insertion. For example, you could use the 5′ UTR and 3′ UTR of uidA lacI, if you planned on deleting/replacing uidA the lacI CDS. According to the original paper, these overlapping regions need only be 40–50 bp long, allowing addition to amplified DNA cassette via oligos <60 primers ≤60 bp.
- Transform strain of interest with pKD46 [Para-λ.redred oripSC101ts AmpR] or a similar inducible λ.λ red, temp-sensitive or counterselectable plasmid. Grow plates at 30°C to maintain the temp-sensitive plasmid.
- pKD46 has the phage λ
- recombination system operon of the λ red locus, encoding Exo, Beta, and Gam, controlled by an arabinose-inducible promoter and on temperature-sensitive pSC101 replicon which replicates at 30° but not 42°.
- Make a small-scale electrocompetent cell prep of the pKD46 transformed cells: grow a seed culture at 30°C in 2×YT to OD600≈1. Then induce λλ red with 1–50 mM (0.05–0.7% m/V) arabinose and grow for 1 hr before harvesting the cells and washing two to four times in 4° 10%V/V glycerol, concentrating it into ≈1/100th the original culture volume. You can proceed to the next step and/or freeze these comp cell aliquots.
- Full induction is 0.32%m/V arabinose = 1.6%V/V of 20%m/V arabinose stock; though less, say 1 mM ~ 0.05%m/V , also works.
- The 2×YT and high OD are important. 3 hr pre-induction growth seems to work well if you inoculate the culture with a 30-fold dilution of saturated preculture.
Why Why can't this be done with growth in SOB or LB as many protocols stipulate? Is the λ.red induction or recombination less efficient?
- Transform Electroporate the λ red–induced pKD46 competent cells with the integration cassette (linear dsDNA (as from PCR); allow to recover for one hour at 37°C; plate on LB agar + antibioticint cassette**, and grow at 37–42°C.
** Use single-resistance-copy antibiotic concentrations- Use media with lower antibiotic concentrations suitable for single gene copy (e.g. kan
- 20 or chl
- 10),
- less than multi-copy
- concentrations (e.g. kan50 or chl25
- ).
- Confirm integration of desired sequence by appropriate genotyping PCRs, using a fraction of resuspensions of integrant colonies in 10 µL PBS/LB. Use PCR primers whose product extends from the integration cassette, through the integration homology, into the genomic context.
- Cure the temp-sensitive pKD46
Inoculate the remainder of a PCR-verified integrant colony suspension in 1 mL LB (+ opt. antibioticint cassette) to saturation at 37–42°C37–43°C. 42–43° is best for curing pSC101ts. Then plate on LB agar (+ optional antibioticint cassette), growing at 37–42° 37–43° to obtain single colonies. - Confirm curing of pKD46
To test for lack of growth in +amp medium and to use the corresponding –amp culture to make a frozen stock, inoculate both 1 mL LB amp and 1–2 mL LB (+ opt. antibioticint cassette) with each of a few colonies. If proceeding to remove the resistance marker, you can use some of the –amp culture as a preculture for preparing competent cells, or you can remove some of the culture in log phase for direct use in preparing competent cell (may require increasing the culture volume).- To split a colony for separate inoculations, either resuspend it in a small volume PBS/LB and inoculate using fractions of this resuspension, or resuspend the colony in a combined volume of medium which you split appropriately and then add different antibiotics.
- 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).
Site-specific recombinatorial removal of resistance marker - Make competent cells with of a pKD46-cured cassette integrant colony/cultureisolate, making sure to use single-copy antibiotic concentrations (or none at all).
- The TSS method or even quicker, lower efficiency chemical competent cell prep methods are fine to use. , such as colony-resuspension transformation, wherein a couple colonies are simply resuspended in TSS or CCMB to make competent enough for pure plasmid transformation.
- Transform with pE-FLP [Pcon-FLPFLP oripSC101ts AmpR] with recovery at 30°C. Plate on LB amp and grow at 37°C to obtain colonies. Though typically not needed, you may test for loss of the resistance marker (and plasmid) as in Step 7, but with colonies tested for individual sensitivity to ampicillin (to ensure loss of pE-FLP) and to antibioticint cassette (to ensure cassette excision), reserving some colony cells for preparing a frozen stock.
- Strong, constitutive expression of Flp from pE-FLP should result in 100% resistance cassette excision without a culturing step at 30°C for prolonged plasmid maintenance.
- If using the classical but inferior pCP20[λ.pR-FLP cI857cI857 oripSC101ts AmpR ChlR], transform a cassette integrant with pCP20, selecting on LB amp agar at 30°C. To induce FLP expression and subsequent pCP20 curing, grow a pCP20-transformed colony in LB (no antibiotic) at 37° and replate after a few hours' growth on LB agar (no antibiotic) growing at 42°C. Grow 4–16 colonies at 37–42°C in LB, expecting <5% of colonies to have successfully excised resistance markers. Test individual colonies for loss of all antibiotics via plating tests.
- Optionally confirm integration of desired sequence by appropriate genotyping PCRs, using a fraction of resuspensions of integrant colonies in 10 µL PBS/LB. Use PCR primers targeting the genomic context flanking the integration homologies (not inside the integration homologies) to test for successful removal of the resistance marker / sequence between the FRT sites (leaving a single FRT). You may additionally use PCR primers targeted inside of the resistance cassette to ensure the cassette is indeed no longer in the genome at any locus, possibly silenced or mutated.
References
- Zhang, Y., F. Buchholz, J. P. Muyrers & A. F. Stewart, (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics 20: 123-128.
- Muyrers, J. P., Y. Zhang, G. Testa & A. F. Stewart, (1999) Rapid modification of bacterial artificial chromosomes by ET- recombination. Nucleic Acids Res. 27: 1555-1557.
- Yu, D.; Ellis, H. M. Ellis, et al. (2000). "An efficient recombination system for chromosome engineering in Escherichia coli." .; Lee, E.-C.; Jenkins, N. A.; Copeland, N. G.; Court, D. L. An Efficient Recombination System for Chromosome Engineering in Escherichia Coli. Proceedings of the National Academy of Sciences of the United States of America 2000, 97 (11): 5978-5983, 5978–5983. https://doi.org/10.1073/pnas.100127597.
- Murphy, K. C. , (1998) Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180: 2063-2071.Bacteriophage Lambda Recombination Functions to Promote Gene Replacement in Escherichia Coli. J Bacteriol 1998, 180 (8), 2063–2071. https://doi.org/10.1128/jb.180.8.2063-2071.1998.
- Datsenko, K. A.; Wanner, B. L. One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (12), 6640–6645. https://doi.org/10.1073/pnas.120163297.