Synthetic Biology Parts, Devices and Applications

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4.5 Selecting Deletion Targets 57

In conclusion, genome streamlining is in large part a trial-and-error process.
The large number of genes with unknown functions and the complex interac-
tions of the constituents of the cell make precise a priori assessments difficult,
especially when synergistic effects of serial deletions are considered. Nevertheless,
based on the general considerations and on individual assessments, some gene
categories can be marked as primary targets for deletion.


4.5.2 Primary Deletion Targets


4.5.2.1 Prophages
Strains of E. coli harbor multiple prophages or phage-related elements that may
represent a significant fraction of the genome (typically 3–5%) (Figure 4.1).
Prophages have a long history of coevolution with their host and seem to be well
integrated in the host physiology. Typically, their genes code for integrases,
lysozymes, and phage structural proteins, but they may carry metabolic and
toxin–antitoxin functions as well. Compared with the entire genome, a higher
than average number of prophage genes have no known function [18, 53].
Regarding their effect on the desired cell characteristics, prophages are Janus-
faced. They can stimulate cell growth in certain conditions and can help the host
to cope with a number of adverse conditions; however, under other conditions,
their effect can be reduced growth, increased sensitivity [54], and instability [55].
Although most of the prophages are cryptic, normally unable to excise and
develop infectious particles, some may excise and lyse the host upon stress [56].
Lytic phage development can be fatal for subsequent cultures of non-lysogenic
strains that may be infected and destroyed [57]. Overall, removal of prophages
and phage remnants does not seem to have adverse effects under customary
growth conditions and may promote uniformity and stability of the culture.


4.5.2.2 Insertion Sequences (ISs)
Insertion sequences (ISs) are small mobile genetic elements carrying the mini-
mal genetic information (inverted repeat ends and transposase gene) for their
own genomic insertion [58]. Typically dozens of ISs of several different classes
reside in the genomes of E. coli strains (Figure 4.1). ISs are important agents of
genetic diversity and are responsible for a significant portion of the mutational
load for the cell. While there are well-documented cases when ISs contribute
to adaptation of the cell to specific conditions, they can generally be viewed as
genomic parasites causing genetic instability, especially under stress [59].
From the practical perspective, removal of ISs significantly increases genetic
stability without adverse effects. In fact, there are cases where presence of ISs
prevents stabile cloning of toxic genes by mutagenesis and selection of altered
clones [5].


4.5.2.3 Defense Systems
Common restriction systems of E. coli (hsdMRS, mcrBC, and mrr) and clustered
regularly interspaced short palindromic repeat (CRISPR) systems provide
defense against invasive foreign genetic material [60, 61]. While these systems
are important factors in the interplay of evolutionary forces shaping the genomes,

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