Biology Reference
In-Depth Information
MG408 which blocks pathogenicity, and identification watermarks. The synthesis of
101 starting 5
7 kb cassettes was outsourced to commercial vendors. Cassettes were
assembled via PCR amplification with a primed bacterial artificial chromosome (BAC)
vector, which allowed for recombination in
E. coli
. The process of assembly
and recombination was repeated until quarter-length genome sequences were
generated. These were amplified with a primed yeast artificial chromosome (YAC)
vector for final recombination in
Saccharomyces cerevisiae
. The extracted genome was
sequence confirmed.
Many improvements have been made to genome synthesis. For example, the Venter
Institute has been able to utilize yeast cells to facilitate genome synthesis.
93
Lartigue et al.
first reported the transformation of modified
M. mycoides
genome, YCpMmyc1.1, into yeast
spheroplasts.
94
The authors then used tools exclusive to yeast to engineer the genome,
before transplanting it into
Mycoplasma capricolum
cells. These tools allow for combinations
of insertions, deletions, and rearrangements that were not previously possible with bacterial
culture techniques. For example, the deletion of the ORF of a nonessential gene, the type III
restriction enzyme, from the genome was performed by introducing a knockout cassette
with a selection marker. Endonuclease-induced double-stranded breaks then promoted
homologous recombination to remove the cassette, enabling seamless gene deletion.
They finally devised a method to avoid restriction endonuclease cleavage of donor DNA in
M. capricolum
through in vitro methylation treatment and inactivation of the recipient
restriction enzyme coding region. Methylation had to be performed in vitro due to
incompatible methylases. The authors avoided problems with gene toxicity because a
difference in stop codon results in truncated gene expression in yeast. Also, transplantation
was limited to bacterial systems of the same genus to ensure compatible cell compositions
between donor and recipient cell types. Despite these notable limitations, the achievement
was groundbreaking. Being able to isolate and transform bacterial genomes into yeast,
engineer them, and retransplant them creates opportunities to manipulate genomes in a
way that was not previously possible, which ultimately results in the assembly of
larger genomes.
95
17
In 2010, a 1.1 Mb bacterial genome was synthesized using an accumulation of assembly
techniques.
95
This was a technical feat, achieving the synthesis of the largest piece of
DNA thus far with the accuracy to drive a functional synthetic organism. Although synthesis
and assembly presented formidable labor and supply costs, the group was able to establish
reliable methods for the assembly of 1 Mb constructs. A five-stage assembly scheme was
used for complete assembly. The authors detail the progression from the assembly of
oligonucleotides to 1078 1-kb cassettes, to 109 10-kb cassettes, to 11 100-kb cassettes,
and to the full 1-Mb genome. Above the 1-kb stage, the authors used the previously
mentioned yeast homologous recombination for assembly. The study was driven by several
modern technologies including fast sequencing and outsourced oligonucleotide synthesis
to commercial companies. The accomplishment was not without roadblocks. One
notable problem was a single base pair deletion that caused a frameshift in
dnaA
,an
essential gene for chromosomal replication. The problem was traced back to the 811
900
assembly, which was corrected through reconstruction and genome engineering in yeast.
Similar problems will continue to present themselves in future genome synthesis projects
and will become technical challenges for researchers in the field. Although the parallel
assembly was efficient, each transition step can introduce random synthesis errors.
Additionally, while homologous recombination in yeast can enable synthetic genome
recombination,
95
it has a 20% efficiency and the underlying mechanisms are still
unknown.
96
Problematic genome fragments with GC-rich noncoding sequences may also be
necessary to discourage yeast repair mechanisms.
97
Finally, fully automating the steps of
genome synthesis and transplantation would ease the labor and time involvement, enabling
synthetic biologists to test hypotheses efficiently.
