by Jason Kelly, MIT
Costs for de novo synthesis of DNA fragments (<10kb) are, and challenges now lie in the assembly of these fragments into ever-larger sequences. One of the main challenges is the fragility of long DNA sequences during the in vitro steps associated with traditional methods for assembling DNA. In a recent publication, Itaya et al (2007) describe a method for assembling 4-6kb DNA fragments in vivo via incorporation in the B. subtilis genome. They demonstrated this homologous recombination-based method by assembling the 134.5 kb rice chloroplast genome from 31 smaller fragments.
The process involves:
- Cloning alternating, overlapping 4-6kb DNA fragments into one of two custom vectors with different selective markers.
- Mixing these vectors sequentially with competent B. subtilis and taking advantage of native homologous recombination to add each fragment to a growing chain within the B. subtilis genome.
- Each new fragment replaces the selective marker added by the previous fragment, allowing the chaining process to continue by switching the antibiotic selection at each step.
- Removal of the fully assembled DNA construct from the genome and re-circularization via previously described methods (Tsuge and Itaya, 2001).
This work-around also demonstrates one method for parallelization of their sequential process. Parallelization provides the speedup necessary for construction of larger DNA segments or genomes. Each addition of a 6kb fragment takes a couple days, so building a synthetic E. coli genome (4.6Mb) through purely serial addition of small fragments would take over four years. A parallelized assembly process combined with Itaya’s previous work (Itaya et al, 2005) incorporating a 3.5Mb natural genome into B. subtilis brings synthetic E. coli-sized genomes closer to reality – will be exciting to watch where this goes.
Note from Thomas: welcome to Jason’s new blog, Free Genes
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