Resources/Plant Synthetic Biology/Plants and iGEM

What is a plant?

The green, multicellular organisms that are considered true plants belong to group of eukaryotes that contain flowering plants, conifers, ferns, hornworts, liverworts, mosses, red algae, green algae and glaucophytes. This group is known as Kingdom Plantae or sometimes Archaeplastidia. The cells of all of the organisms in this group contain intracellular compartments known as plastids, derived from endosymbiosis with cyanobacteria. The genomes of plastids, therefore, are more related to bacteria than to other eukaryotic nuclear genomes. Many genes whose products are active in plastids have moved to the nuclear genome and these gene products are targeted to plastids via an N-terminal peptide known as a Transit Peptide.

Figure 1.Typical Plant Cell Structure. Image in the Public Domain.

Plastids that contain chlorophylls a and b are coloured and allow the organism to photosynthesise, turning solar energy into chemical energy. These plastids are known as chloroplasts. Plastids found in plants that are colourless, for example those found in the cells of endosperm tissues, tubers and roots are known as leucoplasts.

Not all algae belong to the Archaeplastidia. Other algae, including dinoflagellates, golden algae (haptophytes) and brown algae (heterokonts), are found in other kingdoms of the eukaryotic empire. Their host cells and nuclear genomes are only very distantly related to the algae in the Viridaeplantae lineage but their plastids may be much more closely related were generally once free-living green or red algae, acquired by a secondary endosymbiosis.

Any iGEM team that uses a land plant or a eukaryotic algal species from any lineage can self-nominate for the “Excellence in Plant Synthetic Biology” Special award. Like our other Special Prizes, teams will self-nominate their team for this prize on their Judging Form prior to the Giant Jamboree. Details for this new prize are now posted

Plants and Synthetic Biology

Photosynthetic organisms are incredibly important to the planet and to humans. They live on land and in the oceans, covering the planet in vast swathes, visible from space. They convert sunlight into chemical energy providing calories and nutrients. They are also a source of medicines, fibers, construction materials and fuels as well as providing ingredients for a range of consumer products such as paper, adhesives, dyes and resins.

Figure 2. Composite image showing the global distribution of photosynthesis, including both oceanic phytoplankton and terrestrial vegetation. Dark red and blue-green indicate regions of high photosynthetic activity in ocean and land respectively. Image in the public domain, provided by the SeaWiFS Project, Goddard Space Flight Center and ORBIMAGE.

Agriculture is the ultimate large-scale de-centralized production system. In 2012, the Food and Agriculture Organization (The FAO) estimated that cultivation of plants used 40% of earth’s landmass, 70% of its fresh water and employed 30% of the human population. The potential for producing new products in plants is huge.

There are many laboratories and companies that are conducting synthetic biology projects that aim to address important global challenges:
- Providing food security for a growing global population (e.g. The C4 Rice Project, The RIPE project)
- The safe production of therapeutic compounds (e.g. Medicago)
- Reducing human reliance on fossil fuels (e.g. algal biofuels)
- Reducing the contamination of ecosystems with agrochemicals and macronutrients (e.g. ENSA)

Additionally, new roles for plants in our environment are being explored:
- Detectors for specific chemicals e.g sentinel plants
- Cleaning pollutants such as TNT from contaminated land.

How to Engineer a Plant

Engineering a plant cell is not dissimilar from engineering a bacterial cell. Typically a genetic circuit is designed and then assembled in a plasmid in a lab-strain of E. coli. The main differences are that the circuit will be built with eukaryotic gene structure and the final plasmid will also need additional features appropriate to the delivery method to the plant cell.

Plasmid Construction

Construction methods that allow parallel assemble of multiple parts in a single step have allowed for faster and more flexible DNA assembly. Type IIS assembly methods, often known as Golden Gate Cloning is one such technique. Modular Cloning (MoClo) and Golden Braid are two GoldenGate plasmid toolkits that both originated in plant labs.

In 2015, three iGEM teams, Cambridge, NRP-UEA and Valencia wrote and submitted BBF RFC 106 and, the same year, the international plant synthetic biology community published a standard common genetic syntax to allow the exchange of interoperable standard parts for plants. At iGEM, standard parts in this syntax are housed in a variant of the pSB1C3 shipping backbone and are known as PhytoBricks.

iGEM teams may use BioBricks RFC10 assembly standard or, alternatively, teams may also use and submit PhytoBricks and use Type IIS mediated assembly toolkit such as MoClo or GoldenBraid for assembly of single and multi gene constructs. You can learn more about making PhytoBricks and assembling them into eukaryotic genes for expression in plant cells on the Phytobricks Page.

Agrobacterium-mediated DNA delivery

The most widely used method to transfer DNA into plant cells is known as “Agrobacterium-mediated delivery”. Agrobacterium tumefaciens is a pathogenic soil bacterium of the family Rhizobiaceae that infects plants by inserting a section of a large tumour-inducing (Ti) plasmid, known as the T-region, into the genome of the plant host. T-regions are defined by ~25 base pair T-DNA border sequences.

In the 19070's and 80's scientists realised that cloning foreign DNA into T-regions (between the T-DNA border sequences) resulted in integration of this DNA into the plant genome after infection. Suites of plasmids were made to simplify the process of cloning and also to remove the tumour-inducing genes from the bacterium.

Today most scientists use a so-called "binary vector system" in which genes that mediate the infection of the plant by the Agrobacterium, known as virulence (Vir) genes, are encoded on a large plasmid that does not need to be manipulated. A second, smaller plasmid, known as the binary vector contains the T-regions, as well as an origin of replication for A. tumefaciens and a high-copy origin of replication for E. coli. Gene circuits are assembled in E. coli between the borders that define the T-region. The final construct is transferred to A. tumefaciens and a culture is used to infect plant cells or tissues.

The Golden Gate MoClo and Golden Braid plasmids are suites of binary vectors that can be used for Agrobacterium-mediated transformation of plants.

Direct delivery

Direct delivery to nuclear and plastid genomes an achieved by the direct application of purified DNA to cells. In plants this is complicated by the presence of the thick, protective cellulose cell wall. Plant cell walls and membranes can to be penetrated with the aid of a mechanical DNA-carrier such as gold nanoparticles, delivered with a “gene gun”. This is known as ‘bombardment’ or ‘biolistic particle delivery’. DNA can also be delivered by mixing recipient cells with inorganic fibres such as silicon carbide whiskers.

Alternatively, macerozymes and cellulases can be used to digest the polysaccharide cell wall releasing protoplasts (cells without walls). DNA can be directly delivered to protoplasts using PEG (polyethylene glycol) or electroporation to disrupt the membrane.

For all of these methods, the DNA to be inserted can be delivered as a circular plasmid molecule or the desired sequences can be separated from plasmid backbone with restriction endonucleases or amplified by polymerase chain reaction and delivered as linear molecules, free of the plasmid origin of replication and bacterial selection genes.

Other than a high-copy origin of replication to provide sufficient quantities of DNA, there are no specific requirements for the backbones of plasmid vectors in which synthetic genes or transgenes are cloned for these direct delivery methods.

Integration into plastid genomes is typically achieved though homologous recombination and therefore the delivered genes are flanked by regions of homology.

Plant Projects at iGEM

A number of iGEM teams have used plants as a chassis for engineering in the past. You can find information on many of these teams and the parts that they made on the Plant Collections Page. These projects are also a good source of protocols and ideas.

Figure 3. Nicotiana benthamiana leaves under UV light following infiltration (red circles) with Agrobacterium tumefaciens carrying plasmids encoding PROM-AtPDF1.2_CDS-GFP:TERM-AtuOcs. Leaf (B) has been treated with Methyl Jasmonate whilst the control, leaf (A), was untreated. Image by NRP-UEA iGEM 2014

Which plant chassis?

The long regeneration of some plant species can make testing the performance of new genetic circuits problematic. Typically it can take several months to obtain a stably transformed plant - a plant regenerated from a cell in which a gene was integrated and therefore containing the integrated circuit in every cell.

Stable transformation will be difficult for iGEM projects as you will be unable to get though multiple iterations of the design-build-test cycle for your circuits. We recommend the use of a transient system where results can be achieved in just a few days.

The most commonly used systems are transient transfection of leaves of Nicotiana benthamiana (a relative of tobacco from Northern Australia) and protoplasts from tobacco (Nicotiana tabacum) or thale cress (Arabidopsis thaliana) or maize (Zea mays).

The Sheen lab webpage is an excellent source of information and protocols for working with protoplasts.

A protocol for transient transfection of Nicotiana benthamiana is available at Bio-protocols and a video of the leaf infiltration process is available from the Kamoun Lab.

Figure 4.Infiltration of Nicotiana benthamiana leaves. Image by Chandres via Wikimedia. Shared under CC BY 3.0."