In Vivo Recombineering
The in vivo approach requires the transformation into yeast of two or more linear DNA molecules that share unique common end sequences. End-homology can even be provided in trans by overlapping oligonucleotides. If one of the molecules harbors replication and selection elements for yeast and E. coli, then circular episomes up to 110 kilo base-pairs (kbp) in length can be assembled in yeast and transferred to E. coli for downstream applications.
For this purpose a new vector, pYES1L, was constructed (Figure 1A). The plasmid contains a S. cerevisiae’s centromere and the trp1 gene for selection in yeast. In addition F´ ori, the spectinomycin resistance gene, an oriT, and the bacteriophage λ COS site were added for manipulation in E. coli. Rare restriction sites were added for cloning and mapping. We also designed a cassette to convert any E. coli plasmid into a YAC episome, with yeast features similar to pYES1L plus the URA3 gene as an optional counter-selectable marker (Figure 2B).
The system was tested by simultaneously assembling up to 10 linear fragments in a predetermined order into pYES1L (Figure 1C). Adjacent DNA fragments, which shared 30 to 80 base pairs (bp) of end-homology, had been previously cloned into pACYC184 or directly amplified by PCR.
DNA (up to 200 ng) was combined with S. cerevisiae MaV203 competent cells (trp1-901; URA3) and plated onto tryptophan-free CMS agar medium. Clones were verified by colony-PCR and sequencing and/or transferred back to E. coli by directly electroporating colony lysates for restriction profiling. Cloning efficiencies ranged from 58 to 100%.
The approach has also been applied to recombine fragments that do not share end-homology. Here, homology is provided in trans by complementary oligonucleotides that overlap both fragments (Figure 1D). The method is advantageous for re-using fragments in a new sequence context, for cloning DNA targets that cannot be readily amplified by PCR, or for editing the fragment junctions generating end imperfections.