4.5 Selecting Deletion Targets 55Rational, serial construction of targeted genomic deletions requires the full
knowledge of the genome sequence, high quality gene annotations, sufficiently
deep knowledge of cellular physiology, and adequate engineering tools. All these
prerequisites are fulfilled for commonly used E. coli strains. Targeted, rational
design has several advantages: there is no deletion size constraint per se, no sub-
sequent identification of the modifications is required, and optimal serial strat-
egy can be devised (subdivisions of deletions can be made and subsequently
merged). Significantly, the process can be controlled at every step: in case a dele-
tion causes an undesired effect (e.g., loss of fitness), the actual step can be
skipped. On the other hand, the targeted approach suffers from historical contin-
gency: cells with only predesigned deletions, introduced in an order of limited
variability, are being created and tested.
4.5 Selecting Deletion Targets
4.5.1 General Considerations
It is not a trivial task to rationally select dispensable portions of the genome.
The goal is to obtain a streamlined genome that still supports robust and rapid
growth on a range of customary substrates. Usefulness of a gene, obviously, is
context dependent, and our knowledge of the cellular and molecular network
responses under dynamically changing environmental conditions is very limited.
However, there are some gene categories that most likely represent negligible
contribution to fitness under most conditions. There are several approaches
that help identifying these targets.
4.5.1.1 Naturally Evolved Minimal Genomes
The small genomes of obligate symbionts and parasites can provide a template
for a basic set of genes needed for maintaining cellular life. However, simply tak-
ing them as a blueprint for a simple organism can be misleading. Since essential
nutrients and protection are usually provided by the host, the 400–600 genes
they typically harbor are not sufficient to maintain life [13].
4.5.1.2 Gene Essentiality Studies
In most free-living organisms investigated, essential genes make up 10–30% of
the genome. For E. coli, there are several large-scale gene essentiality studies
available. High-throughput random transposon mutagenesis [44] or systematic
gene inactivations [45] were applied to determine the subset of genes, which are
indispensable. However, essentiality studies are not fail-proof. First, essentiality
is a function of the environmental context. Second, both query methods might
miss some hits. Transposon mutagenesis studies assume that a gene, which does
not suffer an insertion event is essential, thus some genes escaping insertion by
chance will be misqualified as essential. Moreover, single or grouped gene inac-
tivations might not reveal redundant, but essential functions, and, conversely,
might identify seemingly essential genes that, in fact, can be deleted in combina-
tion with other genes. Nevertheless, the 295 genes listed as essential candidates