Synthetic biology: From the first synthetic cell to see its current situation and future development

Zhang, LiuYan; Chang, SuHua; Wang, Jing

The synthetic genome was designed based on the genome sequence of M. mycoides. To distinguish the synthetic genome from the natural one, 4 watermark sequences were designed to replace one or more cassettes in regions not interfering with cell viability [1]. The synthetic genome was produced with 1078 of 1080-bp cassettes which covered the whole designed genome. These cassettes were all chemically synthesized, and the ends of each cassette had 80-bp overlaps to adjacent cassettes to facilitate their assembly. A three-stage strategy was utilized to assemble the genome from these cassettes by transformation and homologous recombination in yeast. First, 109 of 10080-bp assemblies were stitched, then 11 of 100000-bp assemblies, and finally the complete genome with 1077947-bp length. The intact synthetic M. mycoides genomes from the yeast were transplanted into the restriction-minus M. capricolum recipient cells by using techniques described in ref. [66] . After culturing and selection, a blue colony of bacteria was found to rapidly grow on a lab plate, which was subsequently identified as the synthetic transplants. Oct. 1995 The whole genome sequencing of Mycoplasma genitalium was completed [30] Dec. 1999 After identified by global transposon mutagenesis, it is estimated that there are about 265-350 genes are essential in the genome of M. genitalium [62] Dec. 2003