Biology Reference
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chemically synthesized. 2 In 2004, a 14 kb ribosomal gene cluster and a 32 kb polyketide
synthase gene cluster were chemically synthesized. In 2008, the 583 kb Mycoplasma
genitalium genome was synthesized at an estimated USD10 million. And in 2010, a 1.1 Mb
Mycoplasma mycoides genome was synthesized at an estimated USD40 million. While the
costs have deterred smaller laboratories from large genome synthesis, gene and genome
synthesis will eventually be integrated into standard bioengineering routines.
Viral genomes are more feasible engineering targets because of their smaller sizes. In 2002,
Cello et al. successfully synthesized a 7.5 kb long poliovirus genome. 89 The genome cDNA
was assembled from 70 mer oligonucleotides to overlapping 400
600 bp fragments and to
three large fragments. These fragments were finally ligated together to form a full genome.
Working on intermediate cassettes allowed for sequence verification and parallelization.
The cDNA was then transcribed into viral RNA using RNA polymerase in cell-free extract.
The resulting genome allowed for translation of viral proteins and replication. Tissue culture
experiments confirmed the pathogenic characteristics of the wild-type poliovirus. The only
modification was the addition of genetic markers.
Smith et al. improved on previous methods of genome synthesis by demonstrating a
method that would only require two weeks. 52 The well-studied 5386 bp PhiX174
bacteriophage genome, which encodes for 11 proteins, was assembled from synthetic
oligonucleotides. This was one of the earliest projects conducted by the Venter Institute in
genome synthesis. Three key modifications to the synthesis protocol were made to improve
quality and decrease time for genome synthesis. One improvement was the addition of a
gel purification step for purchased oligonucleotides in order to eliminate malformed or
truncated oligos. In order to prepare oligonucleotides for PCA, top and bottom strands were
purified separately so that annealing did not occur. The second improvement was the
ligation of oligonucleotides under stringent annealing conditions, which reduced errors.
The final improvement was the assembly of 5
16
6 kb ligation products into a full-length
genome by PCR assembly, which is much faster than traditional restriction digest and
ligation. After PCR amplification and another round of gel purification, functional synthetic
phages were electroporated into E. coli cells and sequenced. Their method contained 9
10
inactivating mutations per synthetic genome, and one sequenced bacteriophage exactly
matched the original sequence. Although most bacteriophages did not match the original
sequence, the team created a reliable and quick method of creating a genome with expected
physical properties.
Viral genome synthesis already has useful applications. There is a demand for effective
influenza vaccines that can be rapidly produced. In recent methods, live viruses can be
attenuated for vaccine development using global codon deoptimization. 90,91 By replacing
codon pairs with suboptimal synonymous pairs in the genome, viral gene product synthesis
is significantly diminished in otherwise functional and replicating viruses. Coleman and
coworkers applied large-scale computer-aided viral genome redesign to perturb genome
codon-pair bias in a process called synthetic attenuated virus engineering (SAVE). 90 This
allows for vaccines to be rapidly designed and synthesized. At the same time, there is a large
safety margin. Reversion to virulence is unlikely because codon-pair deoptimization results
from the accumulated effects of thousands of synonymous mutations. SAVE-attenuated
influenza viruses were created and used for effective vaccination by targeting genes critical
for the assembly and replication of the influenza virus. 91 Further developments in the
predictability of SAVE attenuation and additional assessment of the genetic stability of the
attenuation phenotype may eventually lead to the use of SAVE-attenuated viruses in human
trials. Improvements in DNA synthesis technology can accelerate vaccine development with
the rapid synthesis of custom viral genomes.
In 2008, the synthesis of a 582 970 bp M. genitalium genome called JCVI
1.0 was
2
completed. 92 JCVI
1.0 only differed from the wild-type genome in the exclusion of
2
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