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until our experimental estimations indicated in
Figure 12.3C
. Unsuspected deletions became
obvious when the degree of imbalance exceeded 1000 kbp. Deletions took place in the
guest genome part only, with various sizes up to several hundred kbp, disregarding the right
or left side of BGM. Accommodation of the whole guest genome, consisting of 3500 kbp,
far above the 1000 kbp putative imbalance limit, at one BGM locus was given up. Based on
indiscrimination for the right and left side we were encouraged to investigate how the guest
genome is divided and distributed at both sides of BGM (
Fig. 12.3C
). Amazingly and
expectedly, megacloning of DNA larger than 1000 kbp was achieved by distributing DNAs
into both sides to keep the BGM vector imbalance below 1000-kbp.
3
This empirical and
putative rule was effective enough to produce the CB genome of 7.7 Mbp that possessed
the
Synechocystis
genome (3.5 Mbp) combined with the
B. subtilis
genome (4.2 Mbp). The
CB was the first experimental evidence answering my initial inquiry as to how big the
B. subtilis
genome can become.
25
Can the chimera CB genome function as the
Synechocystis
genome? The present chimera structure for the CB may have disordered the
Synechocystis
genome in terms of
oriC
terC
axis.
26
The origin of DNA replication (
ori
)in
this
Synechocystis
PCC6803 genome has not yet been located (personal communications
from Watanabe and Yoshikawa).
ARE INDIGENOUS PLASIMD GENES ESSENTIAL FOR CELL GROWTH?
The original
Synechocystis
strain PCC6803 carries seven plasmids, four large plasmids, and
three small plasmids.
22
These plasmids, from the smallest (2.4 kbp) to the largest
(120 kbp), summed up about 383 kbp which encoded 397 plasmid-borne genes that
remain to be added to the CB.
3
Those plasmid-borne genes are presently ignored in CB
construction, because of the lack of tools to hold seven plasmids simultaneously in
B. subtilis
.
233
MULTIPLICITY OF BACTERIAL GENOMES
Cells must perform DNA replication and cell division cycles to ensure faithful inheritance of
their genetic materials. The copy number of a bacterial genome per cell in general differs
from species to species.
27
One copy genome of familiar strains
E. coli
K
12 and
B. subtilis
168 is not a typical state among naturally inhabiting eubacteria. The CB strain is likely a
B. subtilis
derivative possessing one genome per cell (Itaya and Yoshikawa, unpublished).
If 10 copies of the genome are essential for
Synechocystis
PCC6803 to grow in a
photosynthetic bacterium medium,
22
the increase of CB genome copy number per cell from
one to 12 should be an absolute requirement prior to gene expression framework. In
contrast to certain genetic regulations on plasmid copy number,
28,29
there is little
understanding about how copy number is regulated. Use of origin sequences from a low
copy plasmid to produce subgenomes in
B. subtilis
could lead to the discovery of a clue to
regulation networks on genome copy number.
30,31
It is worth mentioning here a frequently
asked question whether similar megacloning in a reverse order is possible. Is the
B. subtilis
genome able to be accommodated in the
Synechocystis
genome? Probably, it is less plausible
because integration of DNA into a multicopy genome (
2
10) would be genetically harsh.
It seems a major stumbling block, as we experienced,
27
for the host to accommodate and
maintain 10 chimera genomes stably.
B
SEQUENCE FIDELITY OF GUEST GENOMES IN BGM HOST
Recent high-throughput sequencing technologies unveiled the frequent appearance of single
nucleotide polymorphisms (SNPs) for even variants from laboratory stock cultures.
32
Beneficial alleles can be selected by adaptation in evolution.
33
One may evade this intrinsic
problem by resequencing the DNA, since the time and cost of DNA sequencing became less
of a concern recently. Because the sequence of synthesized genome should be confirmed in
terms of sequence fidelity, it reminded us of the JCVI report on the first synthetic genome.
