Creación de vida sintética

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
Daniel G. Gibson,1 John I. Glass,1 Carole Lartigue,1 Vladimir N. Noskov,1 Ray-Yuan Chuang,1 Mikkel A. Algire,1 Gwynedd A. Benders,2 Michael G. Montague,1 Li Ma,1 Monzia M. Moodie,1 Chuck Merryman,1 Sanjay Vashee,1 Radha Krishnakumar,1 Nacyra Assad-Garcia,1 Cynthia Andrews-Pfannkoch,1 Evgeniya A. Denisova,1 Lei Young,1Zhi-Qing Qi,1 Thomas H. Segall-Shapiro,1 Christopher H. Calvey,1 Prashanth P. Parmar,1 Clyde A. Hutchison III,2 Hamilton O. Smith,2 J. Craig Venter1,2*
The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. 2The J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121, USA.
* 1

To whom correspondence should be addressed. E-mail: jcventer@jcvi.orgcapable of independent growth in the laboratory. More than 100 of the 485 protein-coding genes of M. genitalium are dispensable when disrupted one-at-a-time (4–6). We developed a strategy for assembling viral sized pieces to produce large DNA molecules that enabled us to assemble a synthetic M. genitalium genome in four stages from chemically synthesized DNA cassettes averaging about 6 kb in size.This was accomplished through a combination of in vitro enzymatic methods and in vivo recombination in Saccharomyces cerevisiae. The whole synthetic genome (582,970 bp) was stably grown as a yeast centromeric plasmid (YCp) (7). Several hurdles were overcome in transplanting and expressing a chemically synthesized chromosome in a recipient cell. We needed to improve methods for extracting intactchromosomes from yeast. We also needed to learn how to transplant these genomes into a recipient bacterial cell to establish a cell controlled only by a synthetic genome. Due to the fact that M. genitalium has an extremely slow growth rate, we turned to two faster growing mycoplasma species, M. mycoides subspecies capri (GM12) as donor, and M. capricolum subspecies capricolum (CK) as recipient. Toestablish conditions and procedures for transplanting the synthetic genome out of yeast, we developed methods for cloning entire bacterial chromosomes as centromeric plasmids in yeast, including a native M. mycoides genome (8, 9). However, initial attempts to extract the M. mycoides genome from yeast and transplant it into M. capricolum failed. We discovered that the donor and recipient mycoplasmasshare a common restriction system. The donor genome was methylated in the native M. mycoides cells and was therefore protected against restriction during the transplantation from a native donor cell (10). However, the bacterial genomes grown in yeast are unmethylated and so are not protected from the single restriction system of the recipient cell. We were able to overcome this restriction

Wereport the design, synthesis and assembly of the 1.08Mbp Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a Mycoplasma capricolum recipient cell to create new Mycoplasma mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including “watermark”sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. In 1977, Sanger and colleagues determined the complete genetic code of phage φX174 (1), the first DNA genome to be completely sequenced. Eighteen years later, in 1995, our team was able toread the first complete genetic code of a selfreplicating bacterium, Haemophilus influenzae (2). Reading the genetic code of a wide range of species has increased exponentially from these early studies. Our ability to rapidly digitize genomic information has increased by more than eight orders of magnitude over the past 25 years (3). Efforts to understand all this new genomic information have...