Fig 1.
Schematic view of recombinational cloning and the influence of inserts and vector stoichiometry and quantity.
(A) The plasmid pUC19 was linearized by PCR using the primers pUC3Bf and pUC3Br, which contained short 5′ overhangs (red) matching both ends of the fragment 3B. In parallel, the S. japonicus genomic region 3B was amplified by PCR using the primers 3Bf and 3Br, specifying 5′ short appendages (blue) overlapping the pUC19 vector ends. After gel-purification, vector and fragments were mixed and co-transformed in E. coli. Sequence tracts of 20 bp shared by the vector and fragment were substrates for a homologous recombination reaction resulting in the plasmid p3B. (B) E. coli competent cells were transformed with a fixed amount of 100 ng of the linear pUC19 vector mixed with a variable stoichiometric amount of the fragment 3B. The colony numbers related to each stoichiometric rate represent an average of three independent transformations. (C) Given a fixed insert to vector stoichiometric rate of 2:1, increasing amounts of the pUC19 vector was co-transformed with a proportional quantity of the fragment 3B. The average colony numbers found after each transformation assay (performed in triplicates) were plotted (red). During each cloning assay, competent cells were transformed with the pUC19 vector alone to assess the background of empty vectors (blue).
Fig 2.
The length of overlap between the insert and vector affects cloning yields.
(A) The p3B plasmid was linearized by PCR with primers p3Bf and p3Br. In parallel, the kanMX cassette was amplified by PCR using combinations of the primers kan3Bf and kan3Br containing 5′ overhangs (red tails) varying from 5 to 20 nucleotides identical to the p3B vector. PCR-amplified vector and fragments were gel-purified. Each one of the five different kanMX fragments was independently transformed in E. coli together with the p3B linear plasmid. In vivo homologous recombination between the p3B vector and the kanMX fragments generated the p3Bkan plasmid. (B) The number of colonies obtained increases with the length of overlap between the fragment kanMX and the linear plasmid p3B. Colony numbers represent an average value from three independent transformations. The linear p3B plasmid was transformed alone to assess the background of empty vectors during each transformation. (C) Agarose gels displaying the results of colony PCRs to check for positive insertion events in each one of the five different transformation assays. For each condition, 20 colonies were randomly chosen for PCR amplification with the primers pUC19f and pUC19r. Positive colonies (+) have a molecular weight of 2072 bp. Negative colonies (-) have the molecular weight of 643 bp. Abbreviations correspond to a molecular weight marker (M), a negative control (N) obtained by the PCR amplification of a colony taken from the transformation of the vector alone, and a PCR blank (B) control. Plasmid DNA was extracted from one positive colony and sequenced to confirm the correct insertion event.
Fig 3.
The test of an optimized recombinational cloning protocol.
(A) 0.5 ng of the pUC19 vector was amplified by PCR with the primers dest-f and dest-r. A sequence gap of 398 bp separates the 5′ termini of those primers. Five different templates were amplified with specific primers containing 20 bp long 5′ tails identical to the respective ends of the linear pUC19 vector. The S. japonicus region 6B was amplified by PCR with primers 6Bf and 6Br, while the cassettes zeoMX, natMX, and hphMX were produced with the primers MXf and MXr. Finally, the S. japonicus Ura4 gene was amplified with the primers Ura4f and Ura4r. The vector and PCR fragments were treated with DpnI and co-transformed in E. coli cells. Homologous recombination of each of these fragments with the pUC19 destination vector resulted in a different recombinant plasmid. (B) After transformation of each of the five constructs, 20 colonies were randomly picked to check for positive cloning events (+) by PCR with the primers pUC19f and pUC19r. The sequencing of one plasmid corresponding to each different construct confirmed a positive cloning event. Abbreviations are as described in Fig. 2.
Table 1.
Critical parameters affecting gap-repair cloning efficiency in E. coli.
Fig 4.
The simultaneous cloning of two PCR products into the pUC19 plasmid.
(A) The vector pUC19 was prepared by PCR with the primers dest-f and dest-r. The fragment D1 (red) was generated by PCR amplification of the SjUra4 5′ region with the primers D1f and D1r. The fragment D2 (gray) was generated by PCR amplification of the SjUra4 3′ region with the primers D2f and D2r. Primers were designed to generate a 30 bp overlap between the D1 and D2 fragments, and a 30 bp match of these inserts with the respective termini of the pUC19 vector. Homologous recombination of the D1, D2, and pUC19 fragments generated the plasmid pUraDel, which corresponded to SjUra4 CDS containing a 202 bp internal deletion. (B) PCR screening of 20 randomly chosen colonies with the primers pUC19f and pUC19r. PCR products corresponding to 12 positive colonies (+) had the expected molecular weight of 759 bp. The correct size was confirmed by sequencing a recombinant plasmid extracted from one positive colony. Abbreviations are as described in Fig. 2.
Fig 5.
Homologous recombination between vector and insert generated by restriction endonucleases.
(A) The pNatMX was cleaved with the PvuII endonuclease generating the natMX fragment of 1469 bp. The pUC19 was prepared by digestion with the EcoRI and HindIII restriction enzymes, resulting in a 2639 bp linear plasmid. Homologous recombination between the natMX and pUC19 fragments generated the pUC19Nat plasmid. (B) Agarose gel electrophoresis after gel purification of the fragments natMX and pUC19. (C) The counting of colonies after transformation of the vector alone and co-transformation of the pUC19 plus the fragment natMX. (D) Colony PCR screening confirmed 100% positive cloning events. Abbreviations are as described in Fig. 2.