Biology Reference
In-Depth Information
simplest recipe for plasmid preparation from
E. coli
ever made; just raise the temperature of
growing
E. coli
culture and wait for about 2 hours for
E. coli
to lyse completely.
10
The lysate
was directly mixed with a
B. subtilis
competent cell. Because no biochemical reagents are
used to lyse
E. coli
, no additional cost was paid other than regular incubation facilities.
More valuable, BAC with inserts as large as 100 kbp showed similar high yields and quality
to those purified through traditional biochemical methods.
11
We believe the method should
be a useful tool for the BAC domino method in the near future.
11
It is noteworthy to mention a brief history of lambda lysogen use. After completion of
cloning of the first guest genome, a 48.5-kbp lambda genome in a BGM prototype in
1995,
19,55
my next attempt was to modify the guest lambda DNA directly by lysing
E. coli
cells infected by lambda phages. It led to a discovery not of lambda DNA, but of
stable plasmid DNA in lambda-induced
E. coli
lysate. The finding of stable plasmids in
E.
coli
lysate contradicted the prevailing common sense that degraded
E. coli
cell hold no
complete
E. coli
genome and plasmid. These DNAs should be rapidly degraded because of
no protection mechanism from nucleases abundant in cells. The major nuclease-deficient
mutants of
E. coli
that prolong plasmid stability were investigated to widen available
E. coli
strains as practical tools.
56
A related protocol where prompt lysis by virulent lambda phage
infection allows almost all unmodified
E. coli
transformants to be used, is promising
(Kaneko et al., manuscript in preparation).
Dissection of the Guest Genomes out of the Host Genome
Separation of the guest genome out of the BGM vector is vital to deliver the DNA into the
cell. A method to copy-out the target sequences in BGM and move to
B. subtilis
plasmids
was briefly mentioned earlier and in
Figure 12.4
. Plasmid parts are being improved to carry
larger DNAs.
57
Another method to separate target sequences of the
B. subtilis
genome was
examined.
30,31
As mentioned briefly above and illustrated in
Figure 12.4
, two short identical
sequences are inserted at both ends of the target genome sequence. The
recA
-dependent
intrachromosomal homologous recombination between the short sequences precedes
circularization and physical separation of the intervening genome segment. The separated
segment had to possess an origin of DNA replication (
ori
) different from the chromosomal
oriC
to replicate independently in
B. subtilis
. Although only one case is reported to date,
where the wild-type circular
B. subtilis
genome (4200 kbp) dissected into two circular parts,
300- and 3900-kbp without losing cell viability,
30
future generation of BGM vectors is being
planned to employ the gadget intrinsically. Any parts of guest genomes would be prepared
as dissected DNA in a circular form appropriate to be directly posed into the chassis. In
particular, choice of
ori
from various plasmids with different multiplicity would provide
clues to unveil genome multiplicity.
31
238
Long-Term Storage of Valuable Genome Resources in BGM
Another BGM-related technology less argued is long-term storage of parts or complete forms
of guest genomes. Small DNA can be readily synthesized, and therefore there is no need to
store them. In contrast, reconstructed guest genomes are indispensable because they are not
readily reproduced. Not only complete guest genomes, but also their intermediate DNA
during reconstructions in BGM vectors, are also valuable DNA resources for future
innovations. A BGM vector literally derived from
B. subtilis
is capable of forming spores.
Spores survive for a long time under many hazardous environments including dryness.
B. subtilis
spores start to germinate instantly when exposed in nutrition-rich
environments. Stability of certain guest DNAs inside spores for years is being
confirmed by us (
9
, and our unpublished observations). BGM spores should be
inevitable as a low-cost, long-term reservoir of genomes demanding no special
equipment and facilities.
