In Vitro Recombineering
In a parallel approach we sought to accomplish DNA fragment assembly in vitro where the assembled molecules are directly selected in E. coli. For this purpose we developed a highly efficient enzymatic mix that promotes homologous recombination of up to four DNA fragments plus a vector with 15 bp end-homology. (Note: we were able to prove assembly of up to seven fragments, but the mix is optimized for up to four fragments plus vector).
DNA molecules (20 to 500 ng per fragment in a 2:1 insert:vector molar ratio) were incubated with the enzyme for 30 minutes at room temperature, transformed into TOP10 chemically competent cells, and selected on LB agar plates supplemented with the corresponding antibiotic. Cloning efficiencies ranged from 40% to >90% depending on the number and quality of the DNA fragments (Figure 2A).
Recombination can occur not only at the end of the fragments, but it also works at least up to 32 bp away from their ends (Figure 2B). This attribute is useful for generating cloning variants using a single linearized vector.
The enzyme described above can also be used for recombining and editing the ends of a single DNA molecule, thereby enabling a highly efficient site-directed mutagenesis approach (Figure 3A). Two complementary oligonucleotides with centrally located mutation sites were used.
The DNA methylation and amplification steps were combined into a single reaction. No in vitro digestion or DNA purification was required after methylation or mutagenesis. The system can generate base substitutions, deletions, or insertions of up to 12 nucleotides in plasmids as large as 14 kbp (Figure 3B).