Mycoplasma Laboratorium - Bacterial Chromosome Synthesis

Bacterial Chromosome Synthesis

It is possible to create DNA sequences chemically (Oligonucleotide synthesis) which is achieved by successive rounds of deprotection and coupling of protected phosphoramidite nucleotides with geometrically decreasing yields to length, making sequences longer than 1kb unfeasible. For longer sequences, DNA ligation is required. In 2008 Venter's group published a paper showing that they had managed to create a synthetic genome (a copy of M. mycoides sequence CP001621) by means of a hierarchical strategy. :

• Synthesis ->1kbp: The genome sequence was synthesised by Blue Heron in 1078 1080bp cassettes with 80bp overlap and NotI restriction sites (inefficient but rare cutter).
• Ligation -> 10kbp: 109 Groups of a series of 10 consecutive cassettes were ligatated and cloned in E.coli on a plasmid and the correct permutation checked by sequencing, this would follow a geometric distribution with expected number of trials of 10.
• Multiplex PCR -> 100kbp: 11 Groups of a series of 10 consecutive 10kbp assemblies (grown in yeast) were joined by multiplex PCR, using a primer pair for each 10kbp assembly.
• Isolation and recombination -> secondary assemblies were isolated by means of the plug method above and joining and transformed into yeast spheroplasts without a vector sequence (present in assembly 811-900).