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L. Schorr et al.
cost effectiveness, E. coli is a popular organism for industrial and medical applications, also since desired results are reached with minimal effort [3, 20]. Furthermore,
expanding toolboxes adjusted to E. coli that include genetic elements as promotors,
ribosomal binding sites, or plasmidial origins of replication, ensure an enormous
simplified handling. Human insulin production for instance is nowadays mainly
conducted in E. coli [20]. One approach for the insulin production involves expression of chemically synthesized cDNA encoding for human proinsulin, including
purification steps and excision of C-peptides afterwards. According to the increasing
demand of insulin, optimization in chassis engineering is strongly investigated for
improving the recombinant production [8].
For becoming an advantageous chassis, a selected organism of interest has to be
precisely investigated and optimized. The genome has to be sequenced and genes have
to be exactly annotated. One approach for chassis design is the top-down approach
in which the removal of non-essential genes takes place. The principle is that chassis
designed for specific production purposes contain only essential information for its
function as growing or expressing crucial genes (Fig. 5). The advantage of such
organisms is their functional metabolic network and an increased effectiveness in
synthesizing the product of interest [24]. The design of such chassis can be assisted
by computational analyses, predicting experimental outcomes, and by identifying
the genes which are indispensable for the organism. With a minimal genome chassis,
easier predictions can be made due to the decreased genome complexity [20]. The
new DNA design can be reapplied to the host (e.g. E. coli) and eventual new generated
features of the organism can be investigated [24]. The total synthesis or bottom up of
the genome is another approach representing a milestone in synthetic biology. The
genome of the hepatitis C virus (HCV) was the first completely synthesized genome
in 2002 [26]. In 1995, the minimal genome project under the leadership of Craig
Ventor was initiated with the aim to create an organism with the smallest amount of
genes, the so called minimal genome, that is still able to survive [50]. A minimal
genome has the potential to be the basis for the creation of further chassis. Thus,
it can act as a universal chassis that enables the design of organisms with a higher
complexity that match to specific experimental setups. Mycoplasma genitalium, the
bacterium that Venter investigated, consists of only 525 genes and represents the
smallest known bacterial genome up to now. A de novo synthesized, 1000.000 base
pairs (bp) containing M. mycoides version was created by the transfer into the DNAfree bacterium Mycoplasma capricolum, leading to the novel strain JCVI-syn1.0.
In 2016, the Craig Venter group was able to create the smallest, self-replicating
M. capricolum, JCVI-syn3.0 with 473 genes, that could maintain cellular viability
upon new genetic instructions by indicating the successful progress of the minimal
genome project [26]. Because of natural limitations as host growth rates or biomassproduction properties, it is very difficult to design an optimal host chassis. Vibrio
natriegens, the fastest growing bacterium at lowest nutrients requirements is presently
a promising candidate for an ideal chassis [20]. V. natriegens with a two-fold faster
doubling time as E. coli enables an accelerated cloning and one attempt to create an
optimized chassis with V. natriegens was undertaken by the iGEM team Marburg in
L. Schorr et al.
cost effectiveness, E. coli is a popular organism for industrial and medical applications, also since desired results are reached with minimal effort [3, 20]. Furthermore,
expanding toolboxes adjusted to E. coli that include genetic elements as promotors,
ribosomal binding sites, or plasmidial origins of replication, ensure an enormous
simplified handling. Human insulin production for instance is nowadays mainly
conducted in E. coli [20]. One approach for the insulin production involves expression of chemically synthesized cDNA encoding for human proinsulin, including
purification steps and excision of C-peptides afterwards. According to the increasing
demand of insulin, optimization in chassis engineering is strongly investigated for
improving the recombinant production [8].
For becoming an advantageous chassis, a selected organism of interest has to be
precisely investigated and optimized. The genome has to be sequenced and genes have
to be exactly annotated. One approach for chassis design is the top-down approach
in which the removal of non-essential genes takes place. The principle is that chassis
designed for specific production purposes contain only essential information for its
function as growing or expressing crucial genes (Fig. 5). The advantage of such
organisms is their functional metabolic network and an increased effectiveness in
synthesizing the product of interest [24]. The design of such chassis can be assisted
by computational analyses, predicting experimental outcomes, and by identifying
the genes which are indispensable for the organism. With a minimal genome chassis,
easier predictions can be made due to the decreased genome complexity [20]. The
new DNA design can be reapplied to the host (e.g. E. coli) and eventual new generated
features of the organism can be investigated [24]. The total synthesis or bottom up of
the genome is another approach representing a milestone in synthetic biology. The
genome of the hepatitis C virus (HCV) was the first completely synthesized genome
in 2002 [26]. In 1995, the minimal genome project under the leadership of Craig
Ventor was initiated with the aim to create an organism with the smallest amount of
genes, the so called minimal genome, that is still able to survive [50]. A minimal
genome has the potential to be the basis for the creation of further chassis. Thus,
it can act as a universal chassis that enables the design of organisms with a higher
complexity that match to specific experimental setups. Mycoplasma genitalium, the
bacterium that Venter investigated, consists of only 525 genes and represents the
smallest known bacterial genome up to now. A de novo synthesized, 1000.000 base
pairs (bp) containing M. mycoides version was created by the transfer into the DNAfree bacterium Mycoplasma capricolum, leading to the novel strain JCVI-syn1.0.
In 2016, the Craig Venter group was able to create the smallest, self-replicating
M. capricolum, JCVI-syn3.0 with 473 genes, that could maintain cellular viability
upon new genetic instructions by indicating the successful progress of the minimal
genome project [26]. Because of natural limitations as host growth rates or biomassproduction properties, it is very difficult to design an optimal host chassis. Vibrio
natriegens, the fastest growing bacterium at lowest nutrients requirements is presently
a promising candidate for an ideal chassis [20]. V. natriegens with a two-fold faster
doubling time as E. coli enables an accelerated cloning and one attempt to create an
optimized chassis with V. natriegens was undertaken by the iGEM team Marburg in