2, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Institute of Basic Medical Sciences, Beijing, China
The λ phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner. Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as long as 2 kb with efficiency higher than 1%. Strand bias was consistently observed, and SSOs with sequences identical to the nascent lagging strand during replication always resulted in higher efficiency. Unlike SSO-mediated single nucleotide substitution, homology on each side of SSO flanking the fragment to be deleted was important for successful deletion, and abolishing the host methyl-directed mismatch repair (MMR) system did not lead to detectable changes in deletion efficiency. Finally, we showed that by optimizing its design, SSO-mediated deletion was efficient enough to make it possible to manipulate plasmids without selectable markers.
The ability to direct site-specific modifications on DNA sequence has numerous advantages and applications, especially during the post-genomic era, in which manipulation of large pieces of DNA has become a routine task. The exploitation of phage recombination function serves as an alternative methodology to achieve these purposes and has allowed researchers to overcome limitations endowed by conventional cloning strategy using restriction enzymes and ligase.
Subsequent to its seminal report, recombinogenic engineering using the phage recombination proteins has been well received by numerous laboratories and is documented by the vast amount of literature (1), which also illustrates its versatility for general cloning purposes. Moreover, development of these systems has provided methodologies to suit individual needs. These methods are based either on the rac-encoded RecET system or the bacteriophage-λ Red recombination system (2,3,4,5,6). The RecET-based homologous recombination is mediated by the RecE and RecT proteins, while the Red-based homologous recombination is mediated by three λ-recombination proteins, exo (α), bet (β), and gamma (γ), collectively known as the Red proteins. PCR-generated targeting cassettes have been used to generate deletions and insertions using both systems (2,3,4). In addition, using the λ Red system, it was shown that synthetic single-stranded oligonucleotides (SSOs) may also be used as substrates to generate sequence-specific alterations on DNA molecules with even higher efficiencies. At certain positions on the chromosome, >1% of the SSO-treated cells may be recombinant, making it possible to screen for mutants without using a selectable marker (7,8,9).
Although the exact mechanism of Red-mediated recombination remains elusive, we and others have studied factors affecting efficiency of SSO-mediated single base substitutions, which include length of the oligonucleotide, single-base mismatch that SSO harbors, and its orientation in relation to DNA replication machinery (7, 10,11,12). Besides single nucleotide substitutions, the ability to delete specific DNA sequences from a template is another desired protocol. In an attempt to further advance the utility of phage recombination, we undertook a study to examine factors that are important for deletion of DNA sequences on plasmid DNA. We found that SSO with sequence corresponding to the nascent lagging strand of replication always directed higher efficiency during a deletion process. Importantly, balanced homology of SSO with sequence flanking the fragment to be deleted is required. Additionally, methyl-directed mismatch repair (MMR) is not involved in this process. Finally, we further demonstrated that SSO-mediated deletion of plasmid DNA can be achieved without the use of a selectable marker.Materials and Methods Strains of Bacteria
A modified DH10B strain, called DY380, harboring the red genes under the tight control of the temperature sensitive λ-cI857 repressor has been generated (13). Incubation of DY380 cells at 42°C results in the inactivation of the temperature-sensitive λ repressor and, in turn, allows expression of the Red proteins that catalyze recombination. DY380ΔmutS was generated using recombineering as described previously (11). Genotypes of all bacteria used in this study are listed in (Table 1).Table 1. Genotypes of Bacteria Used in the Study
acro-bioA <> tet indicates substitution of cro-bioA with tet.
bmutS <> kanr indicates substitution of mutS gene with kanr.
To generate the insertion mutant reporters (∼2-kb insertion), a PCR-amplified frt-kanamycin-frt cassette using oligonucleotides kCMF and kCMB with homology to the sequence of the chloramphenicol acetyltransferase (CAT) gene was recombined into the CAT gene carried on pBlue-scriptIIKS(+) plasmid and pBluescriptIISK(+) plasmid to give p(+)mCMKan and p(−)mCMKan, respectively. One base pair of the CAT gene was also deleted during insertion. Subsequent removal of the kanamycin gene using the 294-Flp strain (courtesy of Francis Stewart, The European Molecular Biology Laboratory, Heidelberg, Germany) results in two additional reporter plasmids, p(+)mCMfrt and p(−)mCMfrt, both of which carry a ∼100-bp insertion in the CAT coding sequence. Plasmid used for introduction of deletion mutation, pDB220, was described by Bell et al. (14). All oligonucleotides (Proligo Singapore, Singapore) used in this study are listed in (Table 2).