Biology Reference
In-Depth Information
GENOME CLONING USING A BOTTOM-UP APPROACH
Unlike the straightforward top-down cloning approach, the bottom-up approach stems from
the idea that repeated connection of small DNA segments leads to reconstruction of larger
ones. As small DNAs are readily prepared in
E. coli
, an appropriate host that allows repeated
connection and stable maintenance of large DNAs is needed. To this end, two hosts,
B. subtilis
168 and
S. cerevisiae
were developed independently.
B. subtilis
168, a Gram-positive endospore-forming bacterium possessing a 4215 kbp
genome, shown in Figure 12.2, has been demonstrated to accommodate very large DNAs, as
described in the following sections. This remarkable ability was highlighted in 2005 by
stable accommodation of a 3.5 Mbp-long genome from a nonpathogenic unicellular
photosynthetic bacterium
Synechocystis
PCC6803 (3500 kbp).
3
Meanwhile, the J. Craig
Venter Institute (JCVI) produced synthesized genomes designed based on
Mycoplasma
species using yeast as a final host.
4
The
B. subtilis
host case
3,12
is illustrated in
Figure 12.2A
.
Those two groups used different hosts, but shared the bottom-up concept. Target genomes
are called
for cloning. For
both methods, the guest genome was divided into pieces small enough to be readily
prepared by an
E. coli
system. Small pieces designed to share overlap sequences between
adjacent ones are incorporated into the cytoplasm by virtue of DNA transfer mechanisms
shown in
Figure 12.2B,
and connected together as illustrated in
Figure 12.2A
.
'
guest
'
genomes throughout this chapter in comparison to
'
host
'
It should be mentioned that the guest from
Mycoplasma mycoides
synthesized by JCVI in the
host yeast was directly implanted to a closely related strain,
Mycoplasma caplicorum
.
6
Via
genome replacement in the latter species, the resulting colony was the
Mycoplasma
cell
possessing the complete designed and synthesized
M. mycoides
genome. The JCVI
s
achievement, a spectacular biological and technological feat, opened up a gateway to
numerous genome synthesis applications where the functional genomes can be produced
even starting from chemically synthesized DNA oligomers.
4,5
Differences in using
'
227
(A)
Delivery to third host
or
general chassis [V]
Guest genomes
Full set of
domino clone [I]
Genetically retrieved
guest parts [IV]
Host
BGM vectors
(4200 kbp)
Guest genomes in BGM [III]
Domino integration
and progressive
connection [II]
FIGURE 12.2A
Framework of BGM vector system for production of guest genomes. Host BGM vectors are B. subtilis 168 derivative strains
possessing accessory sequences. pBR322 or BAC are accessory sequences to designate the cloning locus catalyzed by
double homologous recombination with domino clones (I) made on the same plasmid.
3,9,12,16,19
(II) is described in the text
and
Figure 12.2B
. Relevant observations in the case for a largest guest genome (III) are detailed in the text. (IV) is argued in
the text and in
Figure 12.4
. (V) is mentioned throughout this chapter with a keyword called chassis. Antibiotic resistance
markers for efficient selection throughout are omitted.
