60 4 Rational Efforts to Streamline the Escherichia coli Genome
In a second recombination event, the exogenous sequences can be excised to
leave a markerless deletion. Repeating the steps, multiple deletions can be cumu-
lated in the cell [13].
A semi-random genome reduction attempt, combining deletions derived
from mapped transposon-inserted genomic libraries, applied site-specific
recombinase systems (Flp/frt, Cre/lox) for the excision step [73]. The proce-
dure, however, leaves a scar, a 34-bp recognition site in the genome, which may
interfere with subsequent rounds of deletions. The problem can be circum-
vented by the use of mutant recognition sites, but the scheme is complex, and
still a scar is left behind. Most large-scale streamlining projects therefore
used improved, general homology-based deletion methods, producing scarless
deletions.
Mutant target sites of site-specific recombinases, preimplanted in the genome,
can also be used to facilitate the exchange of long DNA fragments between an
episome and the chromosome. Using this strategy, a 126 kbp-long chromosomal
segment was replaced with a 72 kbp synthetic DNA cassette carrying three non-
contiguous genomic deletions. The subsequent elimination of the remaining
loxP sites by homologous recombination and introduction of novel mutant loxP
sites can in theory make this somewhat complicated process applicable for
large-scale genome reduction [74].4.6.2 Basic Methods and Strategies
4.6.2.1 Circular DNA-Based Method
Suicide plasmids, replicons multiplying only under permissive conditions,
serve as delivery vehicles for deletion-forming DNA constructs [75, 76]. The
plasmid carries fused homology arms (~0.5–1.0 kb long DNA segments
matching the flanking sequences of the genomic region to be deleted) for the
first recom bination event, an antibiotic resistance gene as a selection marker,
and a gene (usually sacB) allowing counterselection [77] in the second recom-
bination step. Integration into the genome at one side of the planned deletion
occurs via recombination between one of the homology arms and the corre-
sponding chromosomal sequence, catalyzed by RecA. Such co-integrates are
selected by their antibiotic resistance under nonpermissive conditions for
plasmid replication. Next, cells that resolved the co-integrate in a spontane-
ous, second recombination event, are selected applying counterselection pro-
cedures (e.g., permitting growth of only sacB− cells by using sucrose-containing
medium [78, 79]). Outcome of the procedure can be either recovery of wild
type or formation of a scarless deletion. An advanced, more effective version
of the method applies I-SceI cleavage (the enzyme cuts the co-integrate at a
18-bp site [80] found exclusively in the integrated sequence) as a universal
counterselection tool, which, at the same time, stimulates recombination [81]
(Figure 4.3a).