Tools for Genome Synthesis - Synthetic Biology - page 223

BAC-Mediated Large DNA Shuttling between Plasmid and Genome
in BGM
The source of guest DNAs for the BGM vector is not limited to those from eubacteria. In
addition to organelle genomes, existing plant and animal genomes are promising as DNA
resources and have been shown to be widely available. However, whole plant or animal
genomes are too huge to handle as viewed in Figure 12.1 . Instead, the BGM system specific
for relatively small guest DNAs from plant and animal genomes was developed by use of a
prominent E. coli cloning vector, the BAC vector. 38 Indeed, DNA libraries constructed in
BAC from plant and mouse genomes have greatly contributed to sequence determination
and molecular engineering. 39,40 Cloned DNA in BAC that covers long-range genomic
regions, normally 100 200 kb, can be modified by genetic recombination systems in the

original host E. coli . 41 It is already proven that once BAC is transferred into the BGM
system, any type of sequence alterations of the BAC insert is possible by virtue of
intrinsic natural transformation and associated recA -dependent homologous
recombination, as shown in Figure 12.2B . The BAC/BGM manipulation system 9,16,42 44 is

a novel pipeline to produce transgenic animals and promote a shift for potential BAC
users to the BGM system. 44 Existing BAC libraries if transferred to BGM would produce
another DNA resource to generate remarkably engineered DNA. 8 The more the BGM is
used, the more opportunities and fruitful results are expected. This virtuous cycle should
invoke important contemporary issues in a wider range of genome-related
biotechnology. 8

GC Contents of Guests in BGM
Among factors that influence the accommodation of guest genomes into BGM, the content
of guanine and cytosine of guest DNA was inspected. 45 As an extremely high G

C content
DNA, a megaplasmid pTT27, 250 kbp with GC content (68%) from Thermus thermophilus
HB8 was attempted to accommodate at one locus of BGM by the domino method. During
domino elongation of the pTT27 DNA in the BGM vector, stability of the cloned guest as
small as 200 kbp was apparently reduced, which was unexpected based on the case of
Synechocystis as a guest which allows 1000 kbp 44% G

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C content on average. 3,21 The
reduced stability was reflected by frequent random deletions within the cloned pTT27
insert, 45 and high GC content seems likely a sole reason. Replication of high GC DNA
might be ineffective by B. subtilis molecular machinery for DNA replication, supposedly
adapted to its own genome (43% GC on average). The cellular dNTP pool might be too
small to adapt for a new rounds of high GC DNA synthesis. Molecular mechanisms of the
adverse effect of high GC content in the BGM remain to be examined. Alternatively,
megacloning of DNA with high AT content DNA could give some clues. Any limits on the
BGM cloning system should be tested for broader and more versatile applications. Several
guest genomes possessing various GC contents have been chosen to investigate possible
restrictions (Itaya, unpublished).

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BOTTOM-UP APPROACHES FOR DE NOVO GENOME PRODUCTION
Chemically synthesized oligomers are connected to produce longer DNA in test tubes by use
of promising methods, such as Gibson assembly. 46 As chemical synthesis does not require a
template DNA, guest genome synthesis can be expanded to nonexisting ones designed
even from scratch. A primary obstacle seems to be a lack of the ability to draw
complete nucleotide sequence information, and the second obstacle is that of the
delivery method of the synthesized DNA to the appropriate chassis. This oversimplified
scenario was ascertained by a fascinating epoch-making report from JCVI, 6 where a
guest, the modified Mycoplasma micoides genome prepared in the yeast host, was
delivered to a chassis, a closely related species Mycoplasma capricorum . Employment of