Fig 1.
Plasmids of the Landing Pad System.
The helper plasmid, pTKRED, expresses I-SceI (inducible with L-arabinose) and the λ-Red enzymes (inducible with IPTG), and has a temperature sensitive pSC101 origin of replication that can be cured by growth at 42°C. The LP template plasmid, pTKLP, serves as a PCR template for amplification of either the tetA or galK landing pad, and carries a R6Kγ pir+-dependent origin of replication. The donor plasmid, pTKDP, serves as a fragment donor for the integration of large constructs that have been cloned into the purple region of the plasmid, guided by recombination with landing pad LP1 and LP2 sequences, or custom homology regions as described in this report. The sequence sizes given are for pTKLP-tetA and pTKDP-neo; tetA is exactly replaced with galK in pTKLP-galK, and neo is exactly replaced with various antibiotic resistance genes for alternate versions of pTKDP. Small green boxes are I-SceI restriction sites; Landing Pad Regions 1 and 2 are small red boxes labeled LP1 and LP2 respectively.
Fig 2.
tetA counterselection with NiCl2.
Wildtype MG1655 (black circles, solid line) and MG1655 rrnB::tetA (red triangles, dashed line) were grown in RDM + 0.5% v/v glycerol with concentrations of NiCl2 from 0–10 mM, and the effect of NiCl2 quantified by measuring the growth rate (doublings per hour) in exponential growth. Points are the average of three measurements, and error bars are the SD. The growth rate for both strains at high concentrations of NiCl2 where no observable growth was detected is set to zero. Because of the large differential in growth rate, counterselection against the tetA landing pad can be effectively performed at 5–7 mM NiCl2 in K-12 MG1655.
Fig 3.
Exact, antibiotic-free integration.
Agarose gel electrophoresis of colony PCR products using primers that bind to regions flanking each locus (atpI, nth, and ygcE). The first lanes of each show PCR products from wildtype cells (WT). The second lanes of each show the PCR product after the landing pad is inserted (LP). The last lanes of each show PCR products after integration of the lac operon at each locus (INT).
Table 1.
Exact Integration Statistics.
Fig 4.
(A) Strategy for gene fusion in situ. The tetA landing pad is integrated directly between the stop codon and the next base pair by homologous recombination between regions A and B, where the last 3 bp of region A is the stop codon of the targeted gene. The landing pad is then replaced by recombineering and counterselection using NiCl2, removing the stop codon and fusing the two coding sequences together. Homologous recombination between regions A and A’ (identical to A without TAA STOP) and B and B’ (identical to B with new TAA STOP) results in translational fusion. (B) Agarose gel electrophoresis image of colony PCR products verifying fusions to rpoD and hupA, columns 1–2: wildtype; columns 3–4: tetA Landing Pad (LP) integrants; columns 5–9: rpoD::mCherry fusion; columns 10–14: hupA::ECFP fusion. (C) Sequencing results for fusions of rpoD::mCherry and hupA::ECFP. (D) 400X images of wildtype MG1655 (top) and MG1655 rpoD::mCherry hupA::ECFP (bottom) with brightfield (left column), 561 nm laser excitation for mCherry (middle column) and 457 nm laser excitation for ECFP (right column).
Fig 5.
Scarless deletion by intrachromosomal homologous recombination.
(A) Strategy. The target gene is replaced by recombineering using a landing pad amplified using primers containing homology regions A, B, and C. The landing pad is then eliminated by in vivo I-SceI digestion and λ-Red mediated homologous recombination between regions C, followed by counterselection against tetA landing pad retention with 6 mM NiCl2. (B) Verification of the deletion by colony PCR using primers flanking the rrnB operon. Lane 1: wildtype rrnB from MG1655 (WT; ~6 kbp); Lane 2: MG1655 rrnB::tetA landing pad integrant (LP; ~1.6 kbp). Lane 3–10: 8 randomly picked colonies after deletion (270bp). (C) Sequence of the operon rrnB and the sequencing result after deletion. Targeted homology regions are indicated by same color scheme as in (A).
Fig 6.
Scarless deletion by recombineering with oligonucleotides.
(A) Strategy. The target gene is replaced by recombineering with the tetA landing pad. The landing pad is then replaced by recombineering with short, synthesized oligonucleotides and counterselection with 6 mM NiCl2. (B) Agarose gel electrophoresis of colony PCR products verifying deletion of rrnB. Lane 1: Wildtype rrnB (WT); Lane 2: tetA landing pad integrant; Lanes 3–10 8 randomly sampled colonies after deletion. (C) Sequencing result after deletion compared to the original sequence. Targeted homology regions are indicated by the same color scheme as in (A).