Synthetic Biology | State of the Art
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3
State of
the Art
3.1
Overview of paths to synthetic life
3.1.1
Top-down or bottom-up
The behaviour of synthetic biology parts within a particular host has to be defined to render it repeatable
(Kitney and Freemont 2012). Numerous genes are involved in cellular communication while others have been
shown to be non-essential to cell functioning. It was early suggested that it would be possible to reduce
genome complexity to a minimal set of genes able to sustain (under given external conditions) cell life and
reproduction. This idea is exploited in the so-called top-down approach. The bottom-up approach starts
constructing a synthetic cell from scratch: a life-like entity is built by assembling of molecular components.
These can be of biological nature or instead completely ad hoc chemical components. Between these two
approaches is the concept of xenobiology, which aims at the construction of functional alternative nucleic
acids (Porcar and Pereto 2012).
The top-down approach aims at simplifying already reduced cells to get a “chassis” for synthetic biology
devices to be mounted on and has been tried in bacteria and yeast. Non-essential genes – commonly
responsible for adaptation to stress or altered environmental conditions – are removed, as are intergenic
regions. Prerequisite is a thorough analysis of
suitable regions, usually by employing mutants.
For the bottom-up-approach the whole minimal sequence is compiled from scratch, synthesised and
transferred into a suitable cellular casing. After pioneering work in 2007, the approach has been successfully
used in
Mycoplasma genitalium and
Mycoplasma mycoides/
Mycoplasma capricolum (Lartigue et al. 2007;
Gibson et al. 2008; Gibson et al. 2010). Three major research endeavours may be identified: the large scale
synthesis of microbial genomes, the redesign of metabolic pathways (production of desirable compounds), and
the rational design of genetic logic devices from modular DNA parts (Agapakis et al. 2012).
3.1.2
Prerequisites
Techniques to assemble synthetic genomes
DNA synthesis technologies allow creating entire genomes. In synthetic biology, custom-made DNA is used to
build larger DNA segments, and groups of these fragments are pieced together into larger fragments that are
assembled until the desired DNA product is obtained (Montague et al. 2012). There are ligation dependent
methods, like BioBrick™ and Golden Gate, and overlap dependent methods to assemble overlapping
fragments (see Patron (2014) for a review). Two of the most commonly used methods are BioBricks™ (or
standard assembly) and Gibson Assembly®, complemented by other systems like GoldenBraid, which makes
use of GBparts (fragments of DNA with four-nucleotide overhangs) as minimal standard building blocks
(Gibson et al. 2009; Kitney and Freemont 2012; Sarrion-Perdigones et al. 2013). GoldenBraid has been shown
to serve as a modular assembly system in plant synthetic biology (Sarrion-Perdigones et al. 2014). Depending
on the size, a range of methods is available, including
inter alia BglBricks, CPEC (Circular Polymerase Extension
Cloning), Golden Gate, and for larger assemblies sequence-independent overlap methods such as SLIC
(Sequence- and Ligation-Independent Cloning), InFusion™, Clontech™, Gibson Assembly®, SLiCE (Seamless
Ligation Cloning Extract), and CPEC USER (Uracil-specific excision reagent cloning). So far, in particular TAR
(Transformation-Associated Recombination in
Saccharomyces cerevisiae) and Gibson Assembly® have proved
successful. In contrast to the building of new genomes by DNA synthesis and assembly, alternatively
distributed recombineering methods such as MAGE/CAGE and TRMR in
Escherichia coli or Green Monster in
Synthetic Biology | State of the Art
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Saccharomyces cerevisiae are used. To date, these methods are limited to a narrow set of organisms so that a
number of metabolic and biological tools may therefore not be worked on (Montague et al. 2012).
Standardisation
Standardisation is necessary to accurately reproduce synthetic biology devices and systems. However,
concomitantly the full characterisation of parts is necessary, which has currently not yet been reached to a
sufficient extent. Parts usually need to be characterised in a specific genetic or environmental context and do
not function in a predictable manner when taken out of this context. Thus, it will be necessary to solve the
issues of parts characterisation and interoperability by increasing the scope and diversity of tested designs
(Cameron et al. 2014).
Registry of parts
To make use of parts efficiently, the need for a professional registry of parts was identified (Kitney and
Freemont 2012). The registry comprises a database and should include the full characterisation of parts in the
context of suitable hosts. To tackle the storage and assembly of genetic parts, the Registry of Standard
Biological Parts (RSBP) was established, which facilitates the methodological assembly of parts into larger
circuits by storing them in the standardised “BioBrick™” format. A standard computational language (Synthetic
Biology Open Language, SBOL) was subsequently developed to describe parts and designs, and to facilitate
their exchange.
Chassis and minimal genomes
A core undertaking in synthetic biology is the “minimal genome” concept,
i.e. the minimal set of genes
required to allow cellular life, onto which genes can be added and then transplanted into a chassis (Acevedo-
Rocha et al. 2013). The minimal genome contains the simplest possible components to sustain reproduction,
self-maintenance and evolution (Sole et al. 2007; CBD 2014). To develop a core or minimal
chassis the genome
is reduced to a functionally useful set of genes. The result should be a simple, predictable and programmable
chassis that is able to propagate in a safe and controllable manner, including mechanisms preventing
unintended release into the environment and ensuring isolation from other organisms. The minimal genome
allows avoiding potential risks by, e.g., minimising the potential of cells to propagate under natural
environmental conditions and excluding pathogenicity. The aim is to generate simple cellular systems, which
may be used to answer scientific questions concerning the systematic interplay of cellular modules (Esvelt and
Wang 2013).
A chassis derives from a well-known, safe platform cell factory, involving the reconstruction of a completely
synthetic pathway and the alteration of metabolic fluxes (Nielsen and Keasling 2011).
In industrial production a
few fungal and bacterial cell factories are used, e.g.
Saccharomyces cerevisiae for the production of fuels,
Escherichia coli for producing pharmaceuticals, or
Corynebacterium glutamicum for the production of amino
acids.
Escherichia coli, for example, is an ideal test bed for synthetic biology endeavours because of the already
established deep mechanistic understanding of its biology, its ease of genetic manipulation and the relatively
large number of well-studied gene regulatory systems (Cameron et al. 2014).
3.1.3
Platform cell factories
Synthetic biology and metabolic engineering interact to turn living cells into microbial factories used in
industrial biology (“self-regenerating machines”) (Nielsen and Keasling 2011; Agapakis et al. 2012). The aim is
to have a limited number of “platform cell factories” available to produce a wide range of fuels and chemicals.
A major advantage of such platform is that it may be used to insert many different synthetic pathways. For
example, a platform cell factory may be created such that it produces an important precursor metabolite for
many products, e.g. acetyl Co-A for the production of polyketides (antibiotics, anticancer drugs and