Fig 1.
(A) Generation of a designed mutation in three days using DIRex. (Day 1) A culture expressing λ Red is transformed with two PCR products to generate a semi-stable DIRex intermediate containing a selectable and counter selectable cassette. See Fig 2 for more details. (Day 2) Transformants are isolated and colony-purified on selective medium. (Day 3) Colonies growing on selective medium are picked and streaked on counter-selective medium. (Day 4) Nearly 100% of colonies growing on counter selective media contain the designed mutation. (B) Transferring a previously constructed mutation into another strain by generalized transduction. A phage lysate grown on a strain containing a DIRex intermediate is used as donor in the transduction. The steps involved are the same as in (A) except for a transduction instead of a recombineering step on day 1.
Fig 2.
The method is illustrated with an example for generating a precise deletion of a hypothetical gene. (A) Two overlapping “half-cassettes” are generated in separate PCR reactions (which can be run in parallel in the same PCR cycler) using one locus specific long primer “Fp1” or “Rp1” in combination with the cassette specific primers “cat-midR2” or “cat-midF”, respectively. Each PCR fragment contain one copy of the IR (yellow arrow) and DR (light blue arrrow), as well as one of the recombinogenic 5’-homology extensions. The templates (Acatsac1 and Acatsac3) differ in the location and orientation of the IR sequence, which contains the gene encoding the blue chromoprotein AmilCP. (B) The two “half-cassettes” are mixed in equimolar amounts and electroporated into λ Red induced cells. For formation of a functional cat gene recombination has to occur between the recombinogenic ends and the chromosome, as well as in the sequence overlap between the two “half-cassettes”. (C) The structure of the semi-stable DIRex intermediate. (D) The structure of the final deletion after spontaneous excision of the DIRex intermediate (See S3 Fig for a possible mechanism of excision).
Fig 3.
Light blue arrows indicate the location and orientation of DR sequences between which recombination is expected to occur. Sequence segments on the upper strand are labeled with lower case letters a–i, and on the lower strand the complementary sequences are labeled a’–i’. (A) Using DIRex for deleting a gene. (A, i) Two 40 nt regions of recombineering homology (a-b, dark blue; c-d, green) are chosen on either side of the sequence to delete. (A, ii) The “left” oligo (upper strand) is designed with a 40 nt homology extension (composed of segment a–b) followed by 15 nts from the other side of the sequence to delete (segment c), and a 20 nt 3’ primer (P1). (A, iii) The “right” oligo (lower strand) is designed with a 40 nt homology extension (composed of segment d’–c’) and 15 nts from the other side of the sequence to delete (segment b’), and a 20 nt 3’ primer (P1). (A, iv) The resulting DIRex intermediate, with two identical 30 nt DR sequences (b–c), each containing the designed deletion junction. (v) The designed deletion after excision of the DIRex intermediate. (B) Using DIRex for replacing a native sequence with a designed sequence (in this example replacing the promoter PgeneE with another promoter, Px). (B, i) Two 40 nt regions of recombineering homology (e, dark blue; f, green) are chosen on either side of the sequence to replace. (B, ii) The “left” oligo (upper strand) is designed with a 40 nt homology extension (composed of segment e) followed by the sequence to replace it with (in this example a ~30 nt sequence containing a promoter, Px), and a 20 nt 3’ primer (P1). (B, iii) The “right” oligo (lower strand) is designed with a 40 nt homology extension (composed of segment f’) followed by the sequence to replace it with (the reverse complement of Px), and a 20 nt 3’ primer (P1). (B, iv) The resulting DIRex intermediate, with two identical 30 nt DR sequences, each composed of the replacing sequence (Px). (B, v) The designed replacement after excision of the DIRex intermediate. (C) Using DIRex for introducing a point mutation. The desired mutation is marked with an asterisk. (C, i) Two regions of recombineering homology (g-h and h-i) are chosen on either side of the point mutation. (C, ii) The “left” oligo (upper strand) is designed with a 45–50 nt homology extension (composed of segment g, the desired point, and segment h), and a 20 nt 3’ primer (P1). (C, iii) The “right” oligo (lower strand) is designed with a 40 nt homology extension (composed of segment i’–h’), and a 20 nt 3’ primer (P1). The homology extension ends just next to the nucleotide(s) to be changed (C, iv) The resulting DIRex intermediate, with two identical 25–30 nt DR sequences, with the mutation next to the “left” DR sequence. (C, v) The designed mutant after excision of the DIRex intermediate. See S1 Fig for specific examples.
Fig 4.
Excision frequencies increase with increasing DR size.
(A) Assay for precise excision. The three DIRex constructs in hisA were identical except for having 20, 25 or 30 bp DRs (light blue arrows). When the DIRex intermediate is present the hisA gene is interrupted, and the cell is unable to grow in medium lacking histidine (His-). AmilCP confers blue color and SacB causes sensitivity to sucrose. Selection on sucrose selection plates allows only cells lacking a functional sacB gene to grow. If the cassette is excised a functional wild-type copy of hisA is restored and the cells lose the blue color and becomes sucrose resistant. For a more detailed view of the primers used in this experiment, see panel B in S1 Fig. (B) Frequencies of segregants (white) and false positives (blue) in six independent cultures of each of the three constructs.
Fig 5.
Use of DIRex to generate deletions and replacements.
Dark blue and green areas indicate the recombinogenic homology arms and light blue arrows indicate the DRs used for excision. For each construct, the top line shows the wild-type arrangement, the middle line the DIRex intermediate and the bottom line the final mutant. (A) Deletion of the S. enterica ssrA gene, encoding tmRNA. (B) Deletion of the S. enterica gal operon promoter region. (C) Deletion of the S. enterica araC, B, A and D genes. (D) Deletion of the E. coli araFGH operon, encoding a high affinity L-arabinose transporter. (E) Replacement of the native araE promoter with a synthetic promoter (PJ23106) in both S. enterica and E. coli. (F) Replacement of most of the S. enterica galE coding sequence with a lux transcriptional terminator.
Fig 6.
Alternative strategies for generating point mutations with DIRex.
(A) Schematic view of the recombineering used for these tests. The recipient strain carries a L169R mutation in hisA, rendering it His- (but Trp+). The incoming PCR fragments are designed to repair this mutation, making the wanted transformants (after segregation of the cassette) His+ (but Trp-). (B) Constructs with the mutation in the middle of the DRs. The site of the mutation is indicated in both DRs with asterisks. “Leu” and “Arg” below the DRs indicate which allele was present in the corresponding PCR product. In the table to the right, “Leu” indicates the fraction of transformants that only segregated to the L169 (wanted) allele, “Both” the fraction of transformants that segregated to both alleles, and “Arg” the fraction of transformants that only segregated to the R169 (parental) allele. (C) Constructs with the mutation next to the ter proximal DR. “o” and “p” indicate presence of a 5’-hydroxyl or 5’-phosphate, respectively, at the indicated end of the PCR fragment. (D) Constructs with the mutation next to the ori proximal side. “o” and “p” indicate presence of a 5’-hydroxyl or 5’-phosphate, respectively, at the indicated end of the PCR fragment. (E) Varying the distance between the mutation and the cassette (= size of the DR) or between the mutation and the end of the PCR fragment. Numbers below the different “modules” at the end of the PCR fragment indicate the size in bp of those “modules”. Red shaded areas indicate a strategy or combination that resulted in reduced success rate (compared to the other tests in the same series) while the green shaded area indicate a combination that resulted in improved success rate. Numbers above the DNA maps indicate the length (in bp) of the corresponding “module” in experiments where those sizes were kept constant.