It’s the first time a genome the size of a bacterium has chemically been synthesized that’s about 20 times longer than [any DNA molecule] synthesized before.
This is a huge increase in capability. It has broad implications for DNA nanotechnology and synthetic biology.
This means that the Venter Institute is on the brink of sythesizing a new bacterial life.
began first by resequencing the native M. genitalium genome to ensure that the team was starting with an error free sequence. After obtaining this correct version of the native genome, the team specially designed fragments of chemically synthesized DNA to build 101 “cassettes” of 5,000 to 7,000 base pairs of genetic code. As a measure to differentiate the synthetic genome versus the native genome, the team created “watermarks” in the synthetic genome. These are short inserted or substituted sequences that encode information not typically found in nature. Other changes the team made to the synthetic genome included disrupting a gene to block infectivity. To obtain the cassettes the JCVI team worked primarily with the DNA synthesis company Blue Heron Technology, as well as DNA 2.0 and GENEART.
From here, the team devised a five stage assembly process where the cassettes were joined together in subassemblies to make larger and larger pieces that would eventually be combined to build the whole synthetic M. genitalium genome. In the first step, sets of four cassettes were joined to create 25 subassemblies, each about 24,000 base pairs (24kb). These 24kb fragments were cloned into the bacterium Escherichia coli to produce sufficient DNA for the next steps, and for DNA sequence validation.
The next step involved combining three 24kb fragments together to create 8 assembled blocks, each about 72,000 base pairs. These 1/8th fragments of the whole genome were again cloned into E. coli for DNA production and DNA sequencing. Step three involved combining two 1/8th fragments together to produce large fragments approximately 144,000 base pairs or 1/4th of the whole genome.
At this stage the team could not obtain half genome clones in E. coli, so the team experimented with yeast and found that it tolerated the large foreign DNA molecules well, and that they were able to assemble the fragments together by homologous recombination. This process was used to assemble the last cassettes, from 1/4 genome fragments to the final genome of more than 580,000 base pairs. The final chromosome was again sequenced in order to validate the complete accurate chemical structure.
The synthetic M. genitalium has a molecular weight of 360,110 kilodaltons (kDa). Printed in 10 point font, the letters of the M. genitalium JCVI-1.0 genome span 147 pages.
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