94 5 Functional Requirements in the Program and the Cell Chassis for Next-Generation Synthetic Biology
noticing a period should not be taken as particularly significant). Steady random
insertion of genes via horizontal gene transfer will go toward creating a uniform
distribution of the genes that are most important for the cell life, while frequent
deletion will tend to make them cluster together [85]. Another well‐identified
constraint in the genome of fast‐growing bacteria results from the fact that
genes located near the origin of replication will tend to be in higher copy num
ber in the growing cell as compared with genes located near the terminus of
replication [90]. This difference is also reflected in the distribution of codon
biases classes [87]. When long enough, the bacterial chromosome is further
organized into macrodomains that are insulated from one another and are
essential for genome packaging [89, 113–115]. The presence of plasmids or sev
eral chromosomes alters this distribution [116]. Finally, there is a significant
pressure for important genes to be transcribed from the leading replication
strand in order to avoid transcription/replication conflicts [117]. Knowledge of
these organization constraints is essential for optimizing gene placing in SynBio
constructs.
Management of space is further associated with several kinds of functional
structures, exoskeleton and endoskeleton, scaffolds, and contractile proteins
such as actins and myosins in the cytoplasm. How do the corresponding macro
molecules know where and when to go as the cell grows, changes its shape and
eventually divides? In this context, it was revealing to discover that Bacteria and
Archaea were not different from Eukarya, having a variety of structuring pro
teins, often associated with the inner membrane and contributing to the overall
shape and functional properties of the cell [118]. As a common feature, the
prokaryotic and eukaryotic cytoskeleton proteins couple energy requirement,
via adenosine triphosphate (ATP) and/or guanosine triphosphate (GTP) utiliza
tion in active (energy‐requiring) mechanisms to effect structuring functions and
manage movements. The corresponding logic of engineering design has yet to be
uncovered. A family of proteins, the structural maintenance of chromosome
(SMC) proteins, manages chromosome spatial arrangement and replication, at
the expense of energy [119]. As another example of versatile functional design,
membrane protein topology is coupled to functional addressing, with recently
recognized proteins with dual topology [120]. Interestingly, there is a coupling
between genome evolution and these proteins: genes in families containing dual‐
topology candidates occur in genomes either as pairs or as singletons, and gene
pairs encode two oppositely oriented proteins whereas singletons encode dual‐
topology candidates [121].
Finally, getting in and out of the cell is essential: the cell has to manage the
influx of compounds used to construct biomass and create energy. It has also to
dispose of waste. These processes occur at the membrane, using a variety of
structures. Often, the cell has to extract useful compounds from an environment
where they are considerably diluted. This requires an energy‐dependent active
transport that concentrates molecules up to a thousand‐fold or more. This essen
tial engineering process has a trade‐off: if the outside concentration of the com
pound increases suddenly, the influx will build up an unbearable osmotic
pressure that will require coupling modification of the influx molecule and safety
valves in order to prevent the breakup of the membrane [122, 123].