Biology Reference
In-Depth Information
Homologous recombination through transformation shown in Figure 12.2B is a single
shared principle of the huge BGM vector systems. 1,17,18 The first technical breakthrough was
brought about by accommodation of a complete genome back in 1995. 19 The provisional
success came from E. coli bacteriophage lambda genome (48.5 kbp), a very large DNA at the
time, albeit small when compared with current abundant sequence-known bacterial
genomes. All the fundamental ideas and concepts illustrated in Figure 12.2A were
recognized and established in the prototype work. 19 Expansion to larger guest DNAs than
E. coli bacteriophage lambda had to wait until sequence-determined genomes were available
for choice as guest DNA.
BGM vectors are derived from a B. subtilis Marburg 168 strain, the entire genome sequence
of which was reported in 1997. 20 The BGM system does not employ conventional
restriction endonucleases and DNA ligase to connect DNA segments that are essential for
gene cloning in E. coli . On the contrary, connection steps are carried out through recA -
mediated homologous recombination, an inherent and highly efficient property of the host,
as illustrated in Figures 12.2A and 12.2B . Relevant examples of giant DNA production via
the BGM system are described below.
A Guest Genome from Synechocystis PCC6803
The method to connect small DNA segments in the BGM vector was termed megacloning, 3
which literally came from cloning of DNA with a megabase pair (Mbp) size. The genome
from Synechocystis PCC6803 was chosen as the largest guest in the BGM host. As sequence-
known DNA is a minimal requirement for megacloning, Synechocystis PCC6803, a
nonpathogenic safe and sound bacterium among a dozen sequence-known species available
in 1997, was indeed the only choice when my project started. The genome of this
unicellular photosynthetic bacterium belonging to different phylum, and far in the
phylogenic distance from B. subtilis , was attempted to be reconstructed in BGM. The project
took about seven years and resulted in a publication in 2005. 3 Also referring to other
reviews, 1,17 stepwise connection of neighboring fragments is illustrated in Figure 12.2A , and
the assembly steps of larger fragments are visualized in Figure 12.3A . The guest strain
possesses a 3573 kb (3.5 Mbp) genome and seven plasmids with various sizes. 21 The
intrinsic property of the multicopy genome, estimated more than 10 per cell, 22 strongly
indicated that the PCC6803 strain actually carries total DNA larger than 35 Mbp in a single
cell. This notion will be argued in the next section.
229
Issues Associated with Synechocystis Megacloning
Connection of two bacterial genomes in one cell raised a number of issues previously
poorly argued. A Bacillus
Synechocystis chimera, hereafter putatively named as CyanoBacillus
or CB, was cultivated in B. subtilis growth media (Fig. 12.3A). Immediately after the last
DNA segment was connected for completion of megacloning, CB was examined to see
whether it starts to grow in a Synechocystis growth media, complete synthetic media with no
carbon sources as mentioned in Figure 12.3B . Logically, a vast number of genes from the
guest genome should be expressed properly and coordinately, and dominate cellular gene
regulatory networks from that of B. subtilis . Accordingly, a number of factors and
components, including the cell membrane, cell wall, and metabolic state were expected to
be converted from the host ( B. subtilis ) to adapt to the guest ( Synechocystis ) in response to
changes in culture medium. No CB culture or colony has yet been obtained in liquid
medium, or on solid plate for cyanobacteria. 22
We have learned many lessons in my project during and after the construction of CB. They
are: (1) genes expressed that influence the host; (2) exclusivity of ribosomal RNA genes;
(3) balanced genome structure; (4) plasmid-borne genes; (5) copy number of genome per
cell; and (6) deleterious mutations possibly incorporated during reconstruction. It is
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