λ Red Recombineering

Procedure

1. Construct dsDNA integration cassette (no known hard size limit, but probably <5 kb is most efficient). It needs to consist of:
   1. Homology arms: 5′ and 3′ sequences that overlap with target locus sequence flanking the position of insertion. For example, you could use the 5′ UTR and 3′ UTR of lacI, if you planned on deleting/replacing the lacI CDS. According to the original paper, these overlapping regions need only be 40–50 bp long, allowing addition to amplified cassette via primers ≤60 bp.
      For cassettes larger than around 3 kb, much longer homology region around 500 bp are likely needed since it must use a different recombination mechanism.
   2. Selection/screening marker: commonly an antibiotic resistance cassette (that functions at a known, typically lower, c _antibiotic at genomic copy#). A fluorescent protein (gfp/rfp) 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. You may have FRT (Flp recombinase target) sites flanking the marker to allow later removal of the marker with the site-specific Flp recombinase. 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 Chl^R and Kan^R markers can be amplified from the R6Kγ conditional origin plasmids pKD3 and pKD4/pKD14, respectively, often successful with primers that include the 40–50 bp genomic homology. (This is how the Keio collection was made.) Bennett Lab plasmid A66 has an FRT-flanked Tet^R. 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. pKD plasmids have the classical priming sites annotated.
      The antibiotic resistance must be different from the one used to select for the λ red plasmid in step 3.
   3. 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]
   • This step can be done any time before the DNA is needed in step 5.
   • For ssDNA oligo recombination, design recombineering oligonucleotides according to (7) or (9).
2. Prepare low-efficiency competent cells of base strain of interest.
   • The TSS method or a colony-resuspension method are sufficient, wherein a couple colonies are resuspended in TSS or CCMB to make cells competent enough for purified plasmid transformation.
3. Transform base strain with pKD46, pSIM, or a similar inducible λ red, temp-sensitive or counterselectable plasmid. Grow agar at 30°C to maintain the temp-sensitive plasmid.
   • pKD46 [araC P_ara -λ.red ori _pSC101ts Amp^R] ⁽⁵⁾carries λ phage recombination system operon of the, λ red , encoding Exo, Beta, and Gam, controlled by an arabinose-inducible promoter and on temperature-sensitive pSC101 replicon which replicates at 30° but not 42°.
   • pSIM5 and pSIM9 [Chl^R ori_pSC101^ts] ⁽⁷⁾ are similar, but were measured to give ≈3.5× more oligo recombinants with ≈½ the uninduced recombination rate. pSIM19 [Spt^R ori_pSC101^ts] requires 42° λ red induction.
   • pEcCas [Kan^R ori_pSC101 sacB] ⁽⁸⁾ has Cas9 and λ red and sucrose-counterselectable, not temperature-sensitive.
4. Prepare electrocompetent cells of the pKD46/pSIM-transformed cells at a small scale: grow a culture at 30°C in 2×YT to OD₆₀₀≈1. Then induce λ 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.
   • 1 mM ≈ 0.05% _m/V arabinose is technically full induction, but higher amounts like 20 mM ≈ 0.3% arabinose can produce a more uniform induction for pKD46. pSIM's were induced with 1 mM ara ⁽⁷⁾.
   • The original protocol induces with 1 mM ara from inoculation in SOB and harvests at OD 0.6. But supposedly, 2×YT and high OD produce better results (says who?). 3 hr pre-induction growth seems to work well if you inoculate the culture with a 30-fold dilution of saturated preculture.
   • Consider washing the preculture to remove secreted β-lactamase from AmpR, which otherwise quickly destroys ampicillin in the main comp cell prep culture and removes selection condition for pKD46.
   • pSIM19 has a CI-857-repressed λ red, which requires a 15 min 42° induction step before preparing the electrocompetent cells.
5. Electroporate the λ red–induced pKD46 cells with the integration cassette (linear dsDNA). Allow to recover for 1–3 hr at 37°C; plate on LB agar + antibiotic _{int cassette}**, and grow at 37–42°C.
   
   • ** Use agar with lower antibiotic concentrations suitable for single gene copy (e.g. kan²⁰, chl¹⁰, spt²⁰), less than multi-copy concentrations (e.g. kan⁵⁰, chl²⁵, spt⁵⁰).
   • Longer recovery period (3 hr) can better ensure segregation of chromosomes. For shorter recovery (1 hr), continue antibiotic selection of integration cassette while curing pKD46.
6. 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.
7. Cure the temp-sensitive pKD46 / pSIM
   Inoculate the remainder of a PCR-verified integrant colony suspension in 1 mL LB (+ opt. antibiotic_{int cassette}) to saturation at 37–43°C. 42–43° is best for curing temperature-sensitive replicons. Then plate on LB agar (+ optional antibiotic of integration cassette), growing at 37–43° to obtain single colonies.
8. Confirm curing of pKD46 / pSIM
   Curing of pKD46 [Amp^R] or pSIM5/pSIM9 [Chl^R] must be confirmed by testing for absence of growth in +amp/+chl medium, while also growing the same colony in a nonselective culture to save as a frozen stock and optionally prepare for marker removal. To do this, inoculate both selective 0.5–1 mL LB amp or chl and 0.5–1 mL LB (+ opt. antibiotic for int cassette) with each of a few colonies. If proceeding to remove the resistance marker via Flp recombinase, 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 (increase the culture volume accordingly).
   
   • 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 and add amp to one. Deep well 24, 48, or 96-well blocks can help in parallel culturing.
9. 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 λ.red).
   

Summarized in Marionette paper⁽¹¹⁾:
"In preparation for recombineering, cells were transformed with a plasmid containing Ara-inducible λ Red recombination machinery with a temperature-sensitive origin of replication [probably KD46]. 50μl of overnight culture was subcultured in 50ml LB medium and grown at 30 °C, 250 r.p.m. for 2h. 2mM 
Ara was added, and the culture continued to grow at 30 °C, 250 r.p.m. for 3h. The culture was then centrifuged (4,500 g, 4 °C, 10min) and washed with ice-cold 10% glycerol four times, with the fourth resuspension in 200μl 10% glycerol. Recombineering-ready cells were stored at −80 °C until use. For the first insertion, six genes … were Golden Gate assembled using BsaI into an RK6 suicide vector (which needs Pir protein in order to propagate). Pir-expressing E. coli JTK164H [a DIAL strain⁽¹⁰⁾] cells were transformed, and plasmids were purified, verified, and linearized with BpiI leaving homology to the glvC pseudogene. Recombineering-ready E. coli MG1655 cells were electroporated and transformed with gel-purified, linearized inserts. After an outgrowth of 1h at 37 °C, transformations were plated on LB-agar plates+antibiotic (5μg/ml chloramphenicol). Colonies were picked and grown at 37 °C in LB+antibiotic, and the presence of the insert was verified by colony PCR."

Flp-FRT site-specific recombinatorial removal of selection marker

1. Make competent cells of an FRT-flanked cassette integrant isolate cured of the λ red expression plasmid. Use single-copy antibiotic concentrations for the integration cassette (or none at all, if long recovery was used during cassette integration).
   • 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 cells competent enough for purified plasmid transformation.
2. Transform cells with pE-Flp [P_con-FLP  ori _pSC101ts  Amp^R] with recovery at 30°C. Streak on LB amp⁵⁰ agar (½ normal amp conc.) and grow at 30°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 antibiotic _{int 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.
   • Consider growing the next liquid culture at 42–43°C to further ensure curing of temp-sensitive replicon of pE-Flp. Such culture can be used to prepare a frozen stock in 20% glycerol.
   • If using the classical but inferior pCP20 [λ.p _R-FLP  cI857  ori _pSC101ts  Amp^R Chl^R], 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° for a few hours, and replate on LB agar (no antibiotic) growing at 42°C. Grow 8–16 colonies at 37–42°C in LB, expecting <5% of colonies to have successfully excised resistance markers. Test individual colonies for loss of all resistances via plating ±antibiotics.
3. Optionally confirm integration of desired sequence by appropriate genotyping PCRs, using a fraction of resuspensions of colonies in 10 µL PBS/LB.
   1. Use PCR primers targeting the genomic context flanking the integration homologies (not inside the integration homologies) to test for successful "Flp"ing out of integration cassette, leaving a single FRT. The PCR should use an extension time long enough to make the product that would include the full integration cassette. The original strain with the integration cassette before pE-Flp treatment can be used as a positive control of the PCR detecting the cassette.
   2. You may additionally use PCR primers targeted inside the integration cassette (generally inside the resistance marker) to ensure the cassette is indeed no longer in the strain at any locus, possibly silenced, mutated, or misintegrated elsewhere.