Constructing large DNA segments by iterative clone recombination
Methods for constructing large contiguous segments of DNA will be enabling for Synthetic Biology, where the assembly of genes encoding circuits, biosynthetic pathways or even whole microbial organisms is of interest. Currently, invitro approaches to DNA synthesis are adequate for generating DNAs that are up to 10s of kbp in length, and invivo recombination strategies are more suitable for building DNA constructs that are 100kbp or larger. We have developed a vector system for efficient assembly of large DNA molecules by iterative invivo recombination of fosmid clones. Two custom fosmid vectors have been built, pFOSAMP and pFOSKAN, that support antibiotic switching. Using this technique we rebuilt two non-contiguous regions of the Haemophilus influenzae genome as episomes in recombinogenic Escherichia coli host cells. These regions together comprise190kbp, or 10.4% of the H. influenze genome.
recombineering dna synthesis fosmid gene synthesis synthetic genome lambda red