4.6 Targeted Deletion Techniiues 594.5.2.7 Genetic Diversity-Generating Factors
SOS-induced translesion DNA polymerases (polII, polIV, and polV) are major
sources of mutations in the cell [67, 68]. When DNA is damaged, these polymer-
ases rescue cells by bypassing bulky replication blocks and, at the same time,
introduce point mutations in the genome. Whether the repair function or the
generation of genetic diversity is the primary function is still debated. It seems
that in case of moderate stress, alternative repair pathways can cope with the
damage, but translesion polymerases are nevertheless induced and generate
mutations [69, 70]. Deletion of the genes of translesion DNA polymerases is thus
desirable to keep evolvability of the cell at the minimum. Indeed, it was shown
that elimination of the translesion polymerases reduces the mutation rate of
unstressed cells and, more significantly, prevents the increase of the mutation
rate under stress. Engineered constructs, which pose a burden on cell growth
and are therefore prone to deterioration via mutation and selection, can be
maintained at higher fidelity in such a stabilized host [4]. It should be noted,
however, that in case of heavy stress and DNA damage, when more extensive
DNA repair is needed, lack of the translesion polymerases may cause a reduction
in fitness [70].
4.5.2.8 Redundant and Overlapping Functions
There are several redundant or overlapping functions in E. coli, and deletion of
some of them can be attempted presumably without compromising growth and
robustness. Typical examples include DNAses, RNAses, and transport systems.
For instance, quadruple and quintuple mutations of nucleases were applied,
albeit at a fitness cost under certain conditions, in order to increase the stability
of electroporated oligonucleotides, enhancing the efficiency of oligonucleo-
tide-mediated allelic replacement procedures [71, 72].
4.6 Targeted Deletion Techniques
4.6.1 General Considerations
E. coli is usually viewed as one of the most readily amenable organism for genetic
engineering, with an arsenal of genetic engineering tools available. However, not
all E. coli strains can be equally well manipulated by the usual tools. Differences
in restriction and recombination systems, variable transformation efficiency and
antibiotic sensitivity, resistance to transducing phage, and restricted applicability
of the counterselecting sacB–sucrose system are a few examples of potential
obstacles. From the engineering point of view, K-12 derivatives are the best-
suited strains. To date, nearly all serial, large-scale E. coli genome streamlining
projects have been performed in such cell lines.
Construction of targeted, base pair precision deletions is usually based on
homologous recombination of dsDNA. To create a deletion, an engineered DNA
segment, carrying a selection marker and sequences matching the flanking
genomic regions of a desired deletion, is transformed in the cell, where exchange
with the genomic segment takes place, catalyzed by endogenous recombinases.