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I
IV
I
IV
I
(iii)
stage F
3.5 Mb
I
IV
IV
III
II
III
(iii)
stage G
III
oriC
I
IV( Δ rrnB )
ytqB
(4.2 Mb)
proB
leuB
(iii)
terC
II
I+II
IV
IV( Δ rrnB )
III( Δ rrnA )
16S
23S
5S
tRNA
CyanoBacillus
I+II
IV( Δ rrnB )
FIGURE 12.3A
Stage-specific interference of the guest rrn operon. The Synechocystis PCC6803 circular genome in the upper left was
megacloned in the BGM vector middle left. Figure 1 of reference 3 was reproduced to emphasize (i) balanced structures
around oriCterC axis, indicated by dotted lines and (ii) guest ribosomal operons, presence and absence, of BGM clones.
A 5.4 kbp-long ribosomal RNA operon, rrnA and rrnB, sketched in the bottom left has an identical sequence. Intermediate
BGM clone at the stage F was compatible with the presence of blue circled rrnB. Attempts to elongate by a next domino
elongation accompanied large deletions of the guest part already integrated. Pinpointing deletion of the 5.4-kb rrnB indicated
at the stage, indicated by a red circle, allowed the rest of megacloning to be completed to give a CyanoBacillus. The other
rrnA operon, red circled, should be also deleted. Reassessment of the rrn operon exclusiveness, likely dependent on the
amount of guest genes, is described in the text.
230
noteworthy that a host used by JCVI, S. cerevisiae eukaryotic yeast, can provide the guest
genome in a circular form sketched in Figure 12.1 . As long as guest genomes are originated
from eubacteria as is the case for Mycoplasma species, 4,6 and even 1.66 Mb Prochlorococcus
marinus MED4 genome, 5 their gene expression in yeast is not expected and therefore
considered harmless for the yeast growth. In sharp contrast, synthesis/megacloning of a
bacterial guest in BGM caused various unsuspected issues argued below.
EXPRESSION OF GUEST GENES IN BGM HOST
Chassis is a technical term in synthetic biology for a cell envelope that divides inner
solutions from the outer environments. If two complete genomes are forced to be present in
a chassis, which set of genes, from host or guest, should be dominant for the chassis? In the
case of CB, expression profiles for guest genes of Synechocystis (
3000), and host genes of
B
B. subtilis (
4000) of the CB strain have been analyzed by microarray and tiling array. Most
of the Synechocystis- originated genes were suppressed in CB, resulting in about 100 mRNAs
at low abundance, in contrast to normal expressions for genes from B. subtilis . As to the
transcription apparatus, no RNA polymerases and few sigma factors from the guest were
present among those expressed (Nishida, Yoshikawa, Itaya, unpublished). A proteome
analysis revealed that only dozens of translation products from the guest can clearly be
detected but, curiously, they showed little match to the transcripts (Ishihama and Itaya,
unpublished). Despite some transcriptions actually expressed from the guest genes, proteins
from the guest seemed virtually sterile (Itaya, Yoshikawa, and Nishida, unpublished), as
B
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