Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

Carole Lartigue

Carole Lartigue(1,3), Daniel G. Gibson(1), John I. Glass(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(1), Zhi-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 Venter(1,2*).

(1) The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. (2) The J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121, USA. (3) UMR 1090, Université de Bordeaux-INRA 33883 Villenave D'Ornon, France.

At the J. Craig Venter institute, the "Synthetic biology" team has long been interested in defining the minimal set of genes necessary to sustain cellular life and has proposed to chemically synthesize a minimal genome starting from a set of oligonucleotides. This genome would be then transferred to a recipient cell for taking its control by replacing the native genetic information. As a step toward that goal, our team recently reported (Science 2010;329:52-6), the successful design, synthesis and assembly of the 1.0-Mbp Mycoplasma mycoides natural genome starting from a set of more than 1,000 synthetic DNA cassettes. A stepwise strategy was designed to assemble the complete genome in 3 stages by transformation and homologous recombination in yeast. In the first stage, 1-kbp DNA cassettes received from Blue Heron, were taken 10-at-a-time to produce 10 kb assembly intermediates. In the second stage, these 10-kbp intermediates were taken 10-at-a-time to produce eleven ~100-kbp assembly intermediates. In the final stage, all 11 DNA fragments were assembled into a complete synthetic genome. The assembled synthetic genome has been propagated in yeast as a centromeric plasmid and successfully transplanted into restriction-minus Mycoplasma capricolum cells. The new cells have the phenotypic properties expected for M. mycoides and the designed synthetic DNA sequence, including watermark sequences and other designed gene deletions and polymorphisms. The recombinant cells are capable of continuous self-replication in laboratory conditions.

Concerns about unintentional release of synthetic form of life in the environment have been a big focus for JCVI researchers. In our project, a number of measures have been adopted to ensure biological control. Some of them are inherent to the organism we decided to work with. Mycoplamas are sensitive bacteria naturally dependent on numerous nutrients with limited availability, rendering them unable to grow outside of the laboratory. Additionally Mycoplasma use a non-standard genetic code where the codon UGA is encoding tryptophan instead of the usual opal stop codon. The accidental introduction of synthetic DNA in other organisms will produce truncated proteins that will be not functional. Other measures have been designed during the genome construction process as the removal of few genes potentially involved in the pathogenicity of the organism. JCVI have always considered the ethical and societal implication of their work very seriously.

This study is the proof of concept about the ability to start with digitized genetic information, synthesize new DNA and transplant that synthetic DNA into cells replacing all of the existing genetic information, and as a result create new cells controlled only by that synthetic designed DNA.

The combination of manipulating natural/synthetic bacterial genomes in yeast and transplantation of the modified genome into a recipient cell offer new challenging prospect of addressing biological questions at the genome level rather that at the gene level.