Abstract
The 1.1 Mb Mycoplasma mycoides subspecies capri strain GM12 genome sequence can be found in GenBank (accession CP001668). Starting with this sequence, we made a number of design changes. We disrupted or deleted 12 genes and we introduced four watermark sequences that contained coded information uniquely identifying it as our designed sequence. The sequence was partitioned into 1078 cassettes 1,080 bp in length with 80 bp overlaps. These were chemically synthesized and obtained from a commercial source. The genome was assembled in 3 stages by transformation and homologous recombination in yeast. In the first stage, the 1,080 bp cassettes were taken 10-at-a-time to produce 10 kb assembly intermediates. The 10 kb assemblies were grown in E. coli to obtain sufficient DNA for the next stage. In the second stage, the 10 kb intermediates were taken 10-at-a-time to produce eleven ~100 kb assembly intermediates. In the final stage, all 11 DNA fragments were assembled into the complete synthetic genome and propagated in yeast as centromeric plasmid.
The circular synthetic M. mycoides genome DNA was extracted out of yeast using an agarose plug method to avoid breakage. The free DNA was then transformed into a recipient M. capricolum host cell whose genome differed substantially in sequence from our synthetic genome. Following entry of the synthetic genome into the recipient cell, the M capricolum chromosome was replaced by the synthetic genome under selection for a tetracycline resistance marker carried by the synthetic genome. This "transplantation" process resulted in colonies that appeared similar to those of native M. mycoides. Sequencing showed that the new cells contained only the DNA that we had designed with the exception of 8 new single nucleotide polymorphisms, an E. coli transposon insertion, and an 85-bp duplication in non-essential genes that occurred during the assembly process. We estimate that after about thirty generations of growth the new synthetic cells no longer contain any proteins or structural components from the original M capricolum recipient cells.
This work is helping to launch and define the new field of synthetic genomics. We expect many applications of the new technology. We now have the means to design and build a minimal cell that will define the minimal set of instructions necessary for life.
The circular synthetic M. mycoides genome DNA was extracted out of yeast using an agarose plug method to avoid breakage. The free DNA was then transformed into a recipient M. capricolum host cell whose genome differed substantially in sequence from our synthetic genome. Following entry of the synthetic genome into the recipient cell, the M capricolum chromosome was replaced by the synthetic genome under selection for a tetracycline resistance marker carried by the synthetic genome. This "transplantation" process resulted in colonies that appeared similar to those of native M. mycoides. Sequencing showed that the new cells contained only the DNA that we had designed with the exception of 8 new single nucleotide polymorphisms, an E. coli transposon insertion, and an 85-bp duplication in non-essential genes that occurred during the assembly process. We estimate that after about thirty generations of growth the new synthetic cells no longer contain any proteins or structural components from the original M capricolum recipient cells.
This work is helping to launch and define the new field of synthetic genomics. We expect many applications of the new technology. We now have the means to design and build a minimal cell that will define the minimal set of instructions necessary for life.