eXtra Botany
Commentary
Opportunities in plant
synthetic biology
Charis Cook*, Lisa Martin and Ruth Bastow
School of Life Sciences, University of Warwick, Gibbet Hill
Campus, Coventry CV4 7AL, UK
* To whom correspondence should be addressed. E-mail: Charis@
garnetcommunity.org.uk
Journal of Experimental Botany, Vol. 65, No. 8, pp. 1921–1926, 2014
doi:10.1093/jxb/eru013 Advance Access publication 6 February, 2014
Received 20 August 2013; Revised 20 December 2013;
Accepted 7 January 2014
Abstract
Synthetic biology is an emerging field uniting scientists from all disciplines with the aim of designing or redesigning biological processes. Initially, synthetic biology
breakthroughs came from microbiology, chemistry, physics, computer science, materials science, mathematics,
and engineering disciplines. A transition to multicellular
systems is the next logical step for synthetic biologists
and plants will provide an ideal platform for this new
phase of research. This meeting report highlights some
of the exciting plant synthetic biology projects, and tools
and resources, presented and discussed at the 2013
GARNet workshop on plant synthetic biology.
Key words: Methods, plant science, resources, science
policy, synthetic biology, tools.
Introduction
Synthetic biology is a new approach in scientific research but,
in many ways, it is the natural progression of the way modern science has been undertaken for the past two decades. It
is first of all driven by the explorative and inventive nature
of fundamental research, and has the potential for enormous
commercial impact. Secondly, synthetic biology is inherently
inter-disciplinary, cutting across the scientific fields of chemistry, physics, biology, mathematics, engineering, social science,
and computer science, as well as encompassing design, economics, and marketing. These two basic principles of synthetic
biology are in keeping both with the current need to maximize
returns from public funding, and with the cross-cutting nature
of biological research since the dawn of the genomics era.
Synthetic biology hit the UK government’s radar in
2012. The Department of Business Innovation and Skills
(BIS) instigated a roadmap for UK synthetic biology (UK
Synthetic Biology Roadmap Coordination Group, 2012),
a £20 million investment commitment in this area, and the
Synthetic Biology Leadership Council to assess progress,
update recommendations, and shape priorities. In 2013, this
investment translated into two opportunities for collaborative synthetic biology projects: the Research Councils UK
(RCUK) call for proposals for Multi-disciplinary Research
Centres and the ERASynBio call for transnational research
projects across Europe and the US; and the UK enabling tools and technologies development fund from the
UK government funding bodies: the Technology Strategy
Board (TSB), the Biotechnology and Biological Sciences
Research Council (BBSRC), and the Engineering and
Physical Sciences Research Council (EPSRC). In addition,
TSB, BBSRC, and EPSRC awarded £10 million to a new
Innovation and Knowledge Centre based at Imperial College
London. This Centre was opened in July 2013 to facilitate
the transition between synthetic biology research and industrial application.
In this context, GARNet, the Arabidopsis research network, held the second UK workshop on synthetic biology for
the plant sciences at the University of Nottingham on 21–22
May 2013. This followed on from the 4th New Phytologist
workshop in May 2012 (Osbourn et al., 2012), which also
aimed to introduce synthetic biology to UK plant scientists.
Synthetic biology
The most common definition of synthetic biology is the
design and construction of new biological parts, devices,
and systems and the re-design of existing biological systems
(Arkin et al., 2009). For many biologists, this means reprogramming systems from the DNA level up, whilst for other
groups, including the UK’s Synthetic Biology Leadership
Council, it must also include a commercial end-point (Arkin
et al., 2009). From GARNet’s perspective, it is key that fundamental research underpins all of synthetic biology and that
innovative products are a consequence of this.
At a time when Western economies are struggling, synthetic biology is viewed as a potential game-changer for
innovation and industry. As a result, many synthetic biology
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
1922 | Cook et al.
funders worldwide closely monitor the bioethics of synthetic biology research and application. The USA and the
European Commission both have ethical and best practice
guidelines that must be considered when drafting proposals and strictly adhered to throughout the project (The
National Science Federation, 2012; The European Group
on Ethics in Science and New Technologies to the European
Commission, 2009).
As showcased at the GARNet workshop, projects within
the sphere of ‘synthetic biology’ vary greatly in scope. They
start at the molecular scale, for example, the elegant de novo
protein construction kit of coiled-coil peptides (Moutevelis
and Woolfson, 2009; Fletcher et al., 2012) that can be used
to make a growing number of structures including self-assembling cages (Fletcher et al., 2013). At the supramolecular levels, DNA and the design and generation of minimal synthetic
genomes are important goals in synthetic biology. The ambitious Yeast 2.0 project aims to answer some of the fundamental questions underlying genomics, such as the role of introns
and the importance of genome organization (Dymond et al.,
2011), and to provide a predictable host cell for future synthetic biologists.
Methods developed by synthetic biology, in particular
cloning and genome editing techniques, have expanded the
possibilities of traditional genetic and metabolic engineering
(Neumann and Neumann-Staubitz, 2010). The production
of high value products of complexities not previously possible is now a reality. Artemisinin is an anti-malarial that naturally occurs in the plant Artemisia annua but, until this year,
has been unsuitable for mass production. However, the biosynthetic pathway of its precursor artemisinic acid has been
re-constructed in Saccharomyces cerevisiae and is now close
to being marketed (Paddon et al., 2013). Further, as described
below, nitrogen-fixing wheat is a probable goal rather than an
unattainable dream.
Plant synthetic biology
Synthetic biology is likely to be a valuable approach in the
plant biologist’s toolbox to help meet the challenge of providing food, energy, and other materials from limited natural
resources. Plant synthetic biology is key to this application.
It is also the next logical step in the progression of synthetic
biology after the molecule, genome, and isolated cell level
approaches that have been trialled to date: a transition to multicellular systems. As highlighted by workshop attendees in
the discussion groups, plants are the obvious choice for this
‘next chapter’ of synthetic biology. Plant synthetic biologists
have an excellent knowledge base to work with, and their subjects are sessile, self-repairing, and not surrounded by the same
ethical issues that are often encountered in animal research.
One plant synthetic biology project that exemplifies the
potential of plants as platforms for synthetic biology is the
nitrogen-fixing cereals project led by Giles Oldroyd at the
John Innes Centre. Nitrogen is a critical limiting element for
plant growth and development, but use of nitrogen fertilizers is expensive and can have negative environmental impacts.
Oldroyd and colleagues have previously characterized the
pathways that allow some plants, such as legumes, to fix their
nitrogen via a symbiotic relationship with rhizobium bacteria
(Capoen et al., 2011; Oldroyd et al., 2011; Xie et al., 2012).
They are now attempting to synthesize these pathways in
wheat to develop a ‘self-fertilizing’ cereal.
An example of a successfully executed synthetic pathway
in plants is the signal transduction system built by June
Medford and her group at Colorado State University. The
‘sentinel’ plant containing the complete synthetic pathway
acts as a biosensor that de-colours when it detects a specific molecule(s) such as TNT. Development of this sentinel
involved synthesizing and optimizing a bacterial-based signal transduction pathway in plant cells. Importantly, use of
a prokaryotic mechanism has generated a synthetic pathway that is isolated from any endogenous activity, minimizing cross-talk within the plant cellular environment and
maximizing signal detection (Antunes et al., 2006, 2009,
2011).
The two projects described above fit what might be considered goal-oriented synthetic biology: specifically redesigning a plant/crop for a particular aim. Other plant scientists
are considering the bigger picture: exploring and quantifying certain aspects of plants to provide tools and resources
for the wider plant science community to undertake synthetic
biology.
One potential orthogonal ‘toolkit’ for plant synthetic biology may come from recently identified operon-like clusters
(Field et al., 2011; Mugford et al., 2013). In bacteria, novel
antibiotics have been biosynthesized by identification, characterization and rearrangement of gene clusters (Gottelt
et al., 2010; Gomez-Escribano et al., 2012). The discovery
that some plant secondary metabolite genes, such as antimicrobial terpenoids in oat and triterpenes in the legume Lotus
japonicus, are encoded by gene clusters (Krokida et al., 2013;
Mugford et al., 2013) presents the possibility of shuffling
genes within a cluster to generate novel compounds in plants
in the same way as in bacteria.
Despite the advantages of plant models for synthetic biology and their potential uses as outlined above, it remains a fact
that plants are more complicated than existing synthetic biology
models like Saccharomyces cerevisiae. They have complex signalling pathways, many organs, and often have very large genomes.
To try and overcome these limitations and push the boundaries
of synthetic biology in plants, workshop speaker Jim Haseloff
and a group of international collaborators propose the liverwort
Marchantia polymorpha as a new model for plant synthetic biology, due to its streamlined genome architecture (Ohyama et al.,
2009) containing less genetic redundancy (Sasaki et al., 2007)
than Arabidopsis thaliana, its developmental simplicity and easy
cultivation in suspension, agar or on soil. If ‘explorative’ plant
synthetic biology is to become an established field in the UK,
adoption of new systems like Marchantia may be necessary.
Tools for synthetic biology in plants
To flourish, plant synthetic biology will need a suite of associated tools and resources, some of which will be plant-specific,
An overview of flagship research projects and resources for plant synthetic biology | 1923
but the majority are likely to be adapted from other fields. In
fact, one of the underlying principles of synthetic biology is
that a biological ‘part’ should be able to fit into any system
and behave in a predictable way. Although building a plant
or plant pathways from orthogonal parts—a concept often
likened to a child’s Lego kit—is not currently feasible, tools
that can generate synthetic ‘parts’ in plants do exist and a few
examples are outlined below.
Genome assembly
Gibson Isothermal Assembly, presented at the GARNet workshop by Jim Ajioka (University of Cambridge), is a powerful
tool that can be used to assemble several hundred kilobases of
DNA into a single molecule. DNA fragments are prepared for
assembly by the ligation of specific sequences to each end. In
this case, the specific overhangs that frame the DNA fragment
are complementary to the overhang of another DNA piece and,
during assembly, the DNA pieces fit together like a jigsaw puzzle.
As proof of the efficiency and capacity of the method, Gibson
et al. (2009) constructed a DNA molecule as large as 583 kb and
cloned products up to 300 kb in length in Escherichia coli.
Sylvestre Marillonet’s (Leibniz Institute of Plant
Biochemistry) Golden Gate modular cloning system (Engler
et al., 2008) enables highly efficient assembly of multigene
DNA constructs in a single tube using a normal benchtop
thermocycler. It is based on Type IIS restriction endonucleases, commonly BsaI, which are targeted to the correct location by BsaI sites that flank the gene or fragment of interest.
The enzymes recognize the BsaI sites and make a cut next
to them, leaving a specific four base pair overhang on either
strand, as shown in Fig. 1A. It is possible for large numbers of
Fig. 1. (A) Schematic diagram of Type II restriction enzyme activity, with
BsaI as an example. Uni-directional BsaI sites are designed next to specific
sequences, S1 and S2. After digestion, the overhangs are complementary
to one another and the BsaI sites are excised. When the parts are ligated
together, there is no scar. There is no limit to the amount of specific sequences
used to top and tail the ‘parts,’ so this method can be used to build
constructs with many parts, as in (B), if the BsaI sites are designed correctly.
different fragments to be generated and ligated in a single cycle;
all that is needed is for the BsaI sites to be designed correctly
(Fig. 1B). With proper design, a ligation between two stickyends left from BsaI digestion will result in a ‘scarless’ clone.
The Modular Cloning (MoClo; Weber et al., 2011) adaptation of Golden Gate is a hierarchical cloning system that
enables any multigene construct to be cloned. It is a kit-like
method containing a number of modules and vectors to facilitate gene stacking. This eliminates the need for the complex
primer design necessary for cloning the BsaI sites and specific flanking sequences on to every DNA part in preparation
for assembly. A second adaptation, GoldenBraid (SarrionPerdigones et al., 2011), simplifies the method still further by
using only four destination plasmids. This limits the number
of parts it is possible to assemble in one run, but as each destination plasmid has two different Type IIS restriction sites,
they can be re-arranged multiple times (Sarrion-Perdigones
et al., 2011).
Gibson Assembly and the Golden Gate cloning methods,
along with new, fast DNA synthesis platforms, have revolutionized molecular biology, which had previously been limited by restriction enzymes and ligases demanding specificity.
Five years ago, building an entire synthetic biology pathway
would have been considered impossible due to the time scales
involved, but now it is a feasible option.
Genome editing tools
Transcription activator-like effectors (TALEs; and the
related TALE-nucleases), presented at the workshop by coinventor Sebastian Schornack, and clustered regularly interspaced short palindromic repeats (CRISPR) technologies are
genome editing tools developed for and by synthetic biologists. They perform site-specific, double-stranded DNA cleavage to knock out genes, leaving no foreign DNA behind, and
can stimulate homologous recombination or non-homologous end joining.
TALEs are used by some species of the pathogenic proteobacteria genus Xanthomonas to turn on specific host genes
(Schornack et al., 2008). They comprise nuclear localization
signals, a transcriptional activation domain, and a series of
tandem repeats. Two variable residues in each repeat determine the specific DNA sequence the TALE will target (Boch
et al., 2009). TALE nucleases (TALENs) are TALEs fused
to the catalytic domain of a fokI nuclease (Christian et al.,
2010). They cut DNA strands at highly specific locations and
can perform deletions (Fig. 2A) or, if a donor template is
provided, trigger seamless insertion by homologous recombination. In 2012, TALEN technology was used to remove a
section of a TALE binding site in a rice sucrose efflux transporter hijacked by X. oryzae during infection. This subtle
genetic modification, which introduced no foreign DNA at
all, rendered rice plants resistant to X. oryzae (Li et al., 2012).
CRISPR/Cas systems naturally occur in bacteria and
archaea, providing defence memory against invading phages:
CRISPR-associated endonuclease Cas9 is guided to target
genomic sequences, usually key infection genes, by two RNA
molecules. In programmable CRISPR/Cas genome editing
1924 | Cook et al.
technology, specific sequences on a CRISPR RNA molecule
are designed to target a specific location on the genome
(Jinek et al., 2012; Fig. 2B). Application of the CRISPR/Cas
system for genome editing was initially illustrated in prokaryotes (Gasiunas et al., 2012; Jiang et al., 2013) and animal
cells (Hwang et al., 2012; Chang et al., 2013; Cong et al.,
2013). Since the GARNet workshop on Synthetic Biology,
CRISPR/Cas technology has also been demonstrated in
plants (Li et al., 2013; Nekrasov et al., 2013; Shan et al.,
2013).
A synthetic biology expression system
A powerful expression system for synthetic biology applications is the CPMV-HT transient expression system developed at the John Innes Centre by George Lomonossoff. The
system is itself a feat of synthetic biology engineering: the
inventors modified cow-pea mosaic virus (CPMV) RNA-2 to
turn it into a non-viral expression vector that delivers as high
an expression as viral systems (Sainsbury and Lomonossoff,
2008). Unlike traditional transformation, which requires a
timescale of weeks before the desired protein is expressed
in a young plant, extremely high-level expression is seen just
five days after infiltration with the vector. This system enables high-level transient expression of foreign proteins in
tobacco (Vardakou et al., 2012) and, if it can be applied to
other species, may become a useful tool for synthetic biology.
For example, researchers have used this approach to produce
empty CPMV-like vessels capable of carrying heavy metals
or other foreign molecules like medicines, presenting exciting
possibilities in drug delivery (Aljabali et al., 2010).
Synthetic biology resources
As mentioned in several presentations at the GARNet workshop, many synthetic biology techniques including Gibson
Assembly, Golden Gate Cloning, and TALE technology, are
accessible for academic use; extensively described in papers,
websites, and online protocols. The open ethos of the global
synthetic biology community may have been seeded, and
was certainly strengthened, by the iGEM Foundation and its
associated Registry of Parts. iGEM (international Genetically
Engineered Machine) started in 2003 as an annual competition for undergraduate students. Teams work on a synthetic
biology project and the resulting products or tools are added
to the Parts Registry. Entries in subsequent years build on
existing work using the continually expanding tools in the
iGEM Registry rather than starting from scratch. The competition now has divisions for schools and entrepreneurs
as well as undergraduates. Anyone can use the Registry to
deposit and request DNA parts including promoters, primers, ribosome binding sites, and protein domains, for the
cost of postage. The Registry therefore provides excellent
resources to those working in the synthetic biology sphere. In
fact, according to a survey of synthetic biologists (Kahl and
Endy, 2013), iGEM’s Registry of Standard Biological Parts is
the most widely used synthetic biology repository. Addgene,
also accessible to plant scientists, is the third most commonly
used. Like the Parts Registry it is community-based, but small
processing fees ensure the quality and reliability of its parts.
As well as the resources equipping the synthetic biology
community with physical biological parts, there are many
websites providing open source software. One example is
the Infobiotics Workbench, the co-creator of which Natalio
Fig. 2. Schematic diagram comparing (A) TALEN and (B) CRISPR/Cas9 genome editing. TALEN specificity is determined by a series of 36 amino acid
repeats (AAR), each containing two hypervariable amino acids in positions 13 and 14. The nuclease makes a single-stranded break, necessitating the
design and synthesis of two TALENs for one double-stranded break. CRISPR RNA, including a specific sequence, guides the Cas9 nuclease to the right
target, where it makes a double-stranded break.
An overview of flagship research projects and resources for plant synthetic biology | 1925
Krasnagor presented at the GARNet conference; a framework for designing, implementing, and analysing in silico
experiments, including model checking (Blakes et al., 2011).
Software engineers are also building programs for specific
synthetic biology applications. Information extracted from
time-lapse images of Coleochaete scutata cells was used to
build CellModeller, a tool to model cell division in plant tissues and bacterial colonies (Dupuy et al., 2010). Alongside
practical experiments, it is now being tested as a model for the
effect of foreign transcription factors on plant morphogenesis.
In order to standardize the many pieces of software generated for and by synthetic biologists, an international team
including Guy-Bart Stan (Imperial College London) developed the synthetic biology open language (SBOL). SBOL is
an open-source standard for in silico representation of genetic
designs—not a programming language and not language-specific, but a graphical representation of a design.
Conclusions
Synthetic biology is an established approach in scientific
research, particularly in the microbial and chemical fields.
There are notable flagship plant-based synthetic biology
projects and, as the existing tools and resources from other
spheres are either directly applied, or adapted for use in plant
biology, it is expected that the plant synthetic biology community will grow and exploit the potential of this approach
for understanding plant systems and engineering biological
solutions.
Like the field of systems biology that went before, it will
take time for synthetic biology to become an ‘everyday’
research approach in plant science. Funding opportunities for
researchers to explore and build resources in this area (sandpits), alongside a registry of plant parts and the promotion
and generation of standards to enable informal or formal
sharing of parts, will all help to build a cohesive plant synthetic biology community.
We would strongly encourage plant scientists to dip their
toe into the pool of synthetic biology and take advantage of
the increased funding targeted to this area. There are many
opportunities to get involved in synthetic biology; for example, UK university teams regularly enter the iGEM competition and have won recognition in the past. This is a proven
way of discovering more about the synthetic biology mindset
and making valuable connections for future work.
The ‘synthetic plants’ being designed and built by the workshop speakers and mentioned in this article are just the start:
in the future, plant synthetic biologists may achieve greater
things even than plant sentinels and mineral-fixing crops. The
potential impact of synthetic biology in plants is undeniably
huge and should not be ignored.
Acknowledgements
The authors would like to thank workshop sponsors the New Phytologist
Trust, the Journal of Experimental Botany, and the BBSRC. CC, RB, and
LM are funded by BBSRC grant BB/G021481/1.
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