OpenPlant work programme

Second, the development of new tools is contributing to the engineering of new traits in plants: 
Workpackage F: Altered photosynthesis and leaf structure.
Workpackage G: Changes in plant carbohydrate content.
Workpackage H: Engineered pathways for the metabolic engineering of natural products.
Workpackage I: New forms of symbiosis and nitrogen fixation for crop plants.
Workpackage J: Methods for high level production of biomolecules by transient expression.

Third, we facilitate interdisciplinary exchange, outreach, and responsible research and innovation through diverse initiatives:
Workpackage K: Annual funding round to support small-scale interdisciplinary grants.
Workpackage L: Outreach activities, training and tools for open exchange of DNA parts and other reagents in biotechnology.

I) Developing open technologies that underpin systematic approaches to bioengineering of plants

Simple plant systems as testbeds for engineering

The liverworts (or Marchantiophyta) are descendants of the earliest terrestrial plants. The group is characterised by morphological simplicity. Liverworts have been a largely neglected area of plant biology, but show great promise as model plant systems after recent developments in transformation methods, genome characterisation and biotechnology.

Marchantia polymorpha is the best characterised liverwort. It is a thalloid liverwort, forming a body of sheet-like tissues that possess distinct upper and lower surfaces. The upper surface has a modular structure, with repeated cellular units that form simple cell complexes adapted for photosynthesis and gas exchange. Like other Bryophytes, the gametophyte or haploid generation is dominant phase of the life cycle. Marchantia has a global distribution, and is often found as a weed in horticulture. The plants grow vigorously on soil or artificial media. Marchantia plants spontaneously produce clonal vegetative propagules, or gametogenesis can be induced by exposure to far red light. Male and female plants can be sexually crossed to produce spores. The plants are extraordinarily prolific. A single cross can produce millions of propagules in the form of single-cell spores. Spores can be harvested in huge numbers and stored indefinitely in a cold, desiccated state. Each spore can germinate to produce a new plant, and, unlike higher plants, can undergo the entire developmental sequence to produce an adult plant under direct microscopic observation. Sequencing efforts have provided a draft of the ~280Mbp genome. Most of the major gene families present in more advanced plants are represented by a single or few orthologues in Marchantia, meaning that there is low genetic redundancy. The apparent simplicity of genetic networks in liverworts, combined with the growing set of techniques for genetic manipulation, culture and microscopy, are set to make this primitive plant a major new system for analysis and engineering. OpenPlant has adopted Marchantia as a simple testbed for plant synthetic biology.

Phytobricks: Standard DNA parts for plants

With wide support from the international plant science community, we have established a common genetic syntax for exchange of DNA parts for plants, extensible to all eukaryotes (Patron et al. 2015). This common syntax for plant DNA parts is at the core of RFC 106, posted at OpenWetWare, and accepted as an official standard for DNA parts in the iGEM synthetic biology competition. 

The Phytobrick standard is a consolidated and consistent standard for Type IIS restriction endonuclease­ based assembly of DNA parts to make synthetic genes. It is based on the widely used "Golden-Gate"-type standard, and allows highly efficient assembly of multiple standard parts into genes without the need to isolate DNA fragments. A range of existing techniques such as Gibson assembly, Mo­Clo and Golden Braid can be used for higher order multiple-gene assemblies, however we have developed a simple and flexible protocol for assembly of plant vectors, the Loop Assembly technique.

The Phytobrick standard is general, and applicable to all plants, and other eukaryotes. For work with the model plant Marchantia polymorpha, we have produced MarpoDB, which is “gene­centric” database for MarpoDB describes and presents the Marchantia genome from an engineer’s perspective, rather than a geneticist’s. The database handles the Marchantia genome as a collection of parts. This is highly useful for automatically mining new parts, and managing part description, and part characterisation. We think that this break from standard genome database architecture is essential for tackling the refactoring of synthetic plant genomes. MarpoDB also provides a useful container for gene expression data, and we plan to integrate cellular features via Plant Ontology terms.

Website: (http://marpodb.io)

Automated DNA Assembly

As part of a collaboration between the University of Cambridge, Earlham Institute and the Universidad Católica de Chile, Pollak and Federici have devised a new method for gene assembly based on two Type IIS restriction endonuceases, BsaI and SapI. Loop Assembly allows rapid and efficient production of large DNA constructs, is compatible with widely used Level zero (L0) DNA parts such as Phytobricks, and can be easily automated.  

Loop Assembly requires the alternating use of two Type IIS enzymes, Bsa1 (6-base-pair recognition sequence, 4 base overhang) and Sap1 (7 base-pair recognition sequence, 3 base overhang), and two sets of complementary plasmid vectors that allow efficient and ordered construction of 1, 4, 16, 64 gene fragments.

Roboticised construction of plant genes 

Like other "Golden-gate"-based protocols, Loop Assembly does not require purification of individual DNA fragments, side products are recut during the ligation reaction to drive efficient formation of end-products. Loop Assembly is well suited to automation. OpenPlant researchers at the Earlham Institute and Cambridge are developing methods using acoustic-focusing non-contact liquid handling robots, which increases speed and scale of assembly, while reducing consumable costs and allowing reactions to be performed in nanolitre volumes. 

Cell-free systems

II) Engineering of new traits in plants

The development of new foundational tools and parts will directly contribute to the engineering of new traits in plants, and OpenPlant supports a number of projects in Cambridge and Norwich, with efforts to engineer: 

Altered photosynthesis and leaf structure

Plant leaves are biofactories that can accumulate valuable products in a number of discrete compartments both within and between cells. Furthermore, they also fine tune synthetic pathways in response to environmental signals. While significant progress has been made in defining cell specific gene expression in roots, this has not been achieved in leaves. This is a bottleneck in engineering this easily harvested organ, and there is no central repository of genetic modules to facilitate this. We aim to provide a library of elements that can be used to drive expression of both nuclear and plastid encoded genes in specific compartments of specific cells of leaves, and in addition to control that expression over the day­night cycle. 

Carbohydrate engineering

Plants provide unrivalled opportunities for provision of sugars and polysaccharides for biorefining, biofuels, animal feed, food and other industrial uses. The main goal of this workpackage is to improve the quality and increase the yield of target polymers, and to alter their structure for higher value applications. OpenPlant is funding work to build a registry of novel enzymes that could be used to alter plant cell wall polymers such as xylan, mannan and digestible glucans, and to engineering pathways for the synthesis of plant starches, which have important consequences for food quality and industrial use.

Plant production of natural products

Plants produce a rich and diverse array of natural products. These compounds have important ecological functions, providing protection against pests, diseases, ultraviolet­B damage and other environmental stresses. They are also exploited as pharmaceutical drugs, agrochemicals, within the food and drink industry, and for a wide variety of other industrial biotechnology applications. Although plants are potentially a tremendous source of diverse and valuable natural products, identifying the pathways for the synthesis of these compounds is more complicated than in microbes because the genomes are larger and more complex.

Genome sequencing studies have led to the discovery that the genes for natural products pathways are in many cases organised in operon-­like clusters within plant genomes. This makes it possible to access the genes and enzymes of specialised metabolism in plants far more readily. OpenPlant supports work to harness and exploit the metabolic diversity of plants using synthetic biology approaches.

Biological nitrogen fixation

Nitrogen-based fertilisers used worldwide, to improve crop yields. Around 450 megatonnes of nitrogen based fertiliser are produced annually, consuming 3-5% of the world's annual supply of natural gas. Nitrogen fixation is found naturally in highly specific associations between Rhizobia bacteria and legume plants. The transfer of this symbiotic association to cereals is a major strategic challenge in plant biotechnology, with profound potential benefits for sustainable agriculture. 

OpenPlant have initiated an engineering strategy to transfer the recognition of rhizobial bacteria from legumes to cereals, as the first step towards engineering N-­fixing cereal crops. This is a strategically important challenge and this Gates and BBSRC ­funded programme represents one of the most ambitious engineering strategies in plant signalling. Marchantia provides a fantastic platform for testing synthetic biology approaches in engineering symbiosis signalling that is directly linked to a strategic programme in cereals.

Plant virus-based systems for bioproduction

The CPMV­HT technology, and its associated pEAQ vectors (Sainsbury & Lomonossoff, 2008; Sainsbury et al., 2009), developed by George Lomonossoff (JIC) has established a unique position for the UK for rapid transient expression of proteins in plants through Agrobacterium-­mediated infiltration of Nicotiana benthamiana leaves. CPMV­HT is a highly flexible system and will be developed for a range of applications in the field of plant synthetic biology. 

III) Interdisciplinary exchange, outreach, and responsible research and innovation

OpenPlant Forum

Each year, in the last week of July, we organise the OpenPlant Forum. The OpenPlant Forum brings together OpenPlant and international speakers to explore the potential applications of reprogrammed biological systems, and a framework for exploring the wider implications of these potentially disruptive new technologies. Each year, an overarching theme is chosen, and we aim to cover the strategic, technical, and social implications of this topic. Topics include Reprogramming Agriculture and Frugal and Fast Biotech. 

Website: OpenPlant Forum

OpenPlant Fund

Students and researchers from different disciplines and different institutes are able to arrange easy access to modern equipment and resources, to pursue joint projects. For example, researchers from fields of physics and engineering will have direct access to lab space for joint projects with genetically engineered plants and microbes. Further, the centres provide a space for training programmes in multidisciplinary approaches to engineering in biology and workshops for using synthetic biology tools in plant systems. We are expanding interdisciplinary programmes for undergraduate recruitment, iGEM (in Cambridge and Norwich) and developing programmes for construction of open source bioinstrumentation. These initiatives have proved to be an excellent way of initiating interdisciplinary collaborations, training and cross fertilisation of ideas, and we are keen to expand this across the two institutes.

The OpenPlant Fund was established to support seed projects on a competitive basis through the annual distribution of up to twenty small-scale grants following a lightweight application process and public pitching event. The aim of the fund is to promote the development of plant Synthetic Biology as an interdisciplinary field and to facilitate exchange between The University of Cambridge, the John Innes Centre and Earlham Institute for the development of open technologies and responsible innovation in the context of Synthetic Biology. The OpenPlant Fund follows the prototype SRI Fund and the programmes have created over 60 open source projects for synthetic biology. 

Website: OpenPlant Fund

Biomaker Challenge

OpenPlant provides funding for interdisciplinary team-based projects at the intersection of electronics, 3D printing, sensor technology, low cost DIY instrumentation and biology - together with with workshops and outreach events. This OpenPlant-funded Biomaker project aims to build open technologies and promote development of research skills and collaborations. They tap into existing open standards and a rich ecosystem of resources for microcontrollers, first established to simplify programming and physical computing for designers, artists and scientists. These tools allow biologists to program and develop real-world laboratory tools. Further, the Biomaker projects provide a direct route for physical scientists and engineers to get hands-on experience with biological systems.

Website: Biomaker Challenge

Biomakespace

OpenPlant collaborates with the ‘Biomakespace’: an innovation space for biology and biological engineering. This effort builds on the success of Cambridge Makespace, a popular community workshop that caters for over 250 members, making engineering and manufacturing technologies accessible to a wide spectrum of innovators and enthusiasts to develop projects and ideas in an informal setting. Biomakespace complements this existing provision in the city with space for experimental biology and fabrication tools focused on scientific applications. The space aims to:

• Bring together biologists, engineers, technologists and others for meeting and co-working in an informal and extra-institutional setting.

• Support new and existing interdisciplinary collaborations.

• Raise awareness of and skills for synthetic biology, one of the UK’s ‘eight great technologies’.

• Build a cross-disciplinary and cross-sector community for synthetic biology in the city, with a focus on open technology and innovation.

• Provide activities such as training and skills sharing sessions, networking events and foster links with innovation and seed funding schemes and local bioincubator spaces and accelerator programmes.

Website: Biomakespace

SAW Trust

The science, art and writing (SAW) initiative breaks down traditional barriers between the arts and sciences. The SAW Trust provides training in project design to scientists working in collaboration with professional artists and writers who come together as teams to deliver projects themed on the scientists’ research topics. In schools, SAW programmes inspire creative artistic and scientific endeavour. Children realise that science and the arts are interconnected – and they discover new and exciting ways of looking at the world. SAW Trust works closely with OpenPlant researchers to explore themes of plants and biological engineering.

Website: SAW Trust

Responsible Innovation

The social acceptance of genetic modification in field-­grown crop plants remains a significant barrier to the adoption of plant synthetic biology in the UK. OpenPlant research, public engagement and outreach efforts promote (i) models for decentralised ownership and control of key technologies, (ii) use precision gene editing technologies, (iii) development of new crop traits with improved properties, sustainable production, resource management and environmental impact, and (iv) aid international development and technical exchange for agriculture and sustainable land use.

OpenPlant has organised, sponsored and contributed to international workshops on intellectual property (IP), material transfer and responsible innovation. An important part of OpenPlant’s vision is centered around open exchange of tools and techniques:

Sharing materials

Current IP practices and restrictive licensing threaten to restrict innovation as the scale of DNA systems increases. We believe that the field needs to explore new “two-tier” intellectual property models that will protect investment in applications, while promote sharing of DNA components and freedom-to-operate for small companies in commercial applications of Synthetic Biology.

We are collaborating with the Biobricks Foundation on an Open Materials Transfer Agreement (OpenMTA). This is a simple, standardized legal tool that enables individuals and organizations to share their materials and associated data on an open basis. 

The primary purpose of the OMTA is to eliminate or reduce transaction costs associated with access, use, modification, and redistribution of materials and associated data. This in turn will help minimize waste and redundancy in the scientific research process and promote access to materials and associated data for researchers in less privileged institutions and world regions.

Website: OpenMTA