Using Gibson Assembly® to Create the First Synthetic Cell

DNA synthesis and activation of a complete bacterial genome to produce the first synthetic cell

In 2010 colleagues at the J. Craig Venter Institute designed, synthesized, and assembled a 1.08-Mbp Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information. The complete genome was transplanted 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, which included "watermark" sequences and other designed gene deletions. The new cells have expected phenotypic properties and are capable of continuous self-replication. Science. 2010 May 20.

In order to complete the project, Gibson and his colleagues relied on a multistage assembly process starting with oligonucleotides. The Gibson Assembly® method was used in combination with yeast homologous recombination to combine sequence verified DNA intermediates into longer constructs that eventually constituted the entire genome.

SGI-DNA uses similar technologies to assemble large pieces of DNA for its global partners. As part of a high value DNA synthesis service, SGI-DNA works with partners to identify the most cost effective and timely approach to a large DNA synthesis project. SGI-DNA has the ability to preserve and catalogue intermediate pieces of DNA for its customers enabling quick and inexpensive redesign if needed.

Learn more about the synthetic cell here:
Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

Fig. 1
The assembly of a synthetic M. mycoides genome in yeast. A synthetic M. mycoides genome was assembled from 1078 overlapping DNA cassettes in three steps. In the first step, 1080-bp cassettes (orange arrows), produced from overlapping synthetic oligonucleotides, were recombined in sets of 10 to produce 109 ~10-kb assemblies (blue arrows). These were then recombined in sets of 10 to produce 11 ~100-kb assemblies (green arrows). In the final stage of assembly, these 11 fragments were recombined into the complete genome (red circle).

Fig. 1