OpenPlant: Open Tools and Technologies

The OpenPlant initiative supports the development of open technologies that will underpin systematic approaches to bioengineering of plants:

• Development of the lower plant Marchantia as a simple and facile chassis for Synthetic Biology, to enable high throughput screening and analysis at the cellular scale (Workpackage A)

• A common syntax and moderated archive for plant DNA parts and assembly of genetic circuits (Workpackage B)

• New mechanisms for regulation of gene expression (Workpackage C)

• Techniques for routine genome-scale engineering in plants (Workpackage D)

• Software tools with improved performance for DNA part catalogues, automated assembly, modelling of synthetic gene circuits and cellular morphogenesis (Workpackage E)

Open Tools and Technologies at a glance:

Simple plant systems as testbeds for engineering (Workpackage A)

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.

CELL-FREE SYSTEMS

Recent technical advances in the preparation of microbial cell-free extracts have given rise to a new class of highly efficient systems for gene expression that are cheap to deploy and have huge potential benefit for the provision of a wide variety of diagnostics, sensors, vaccines and research materials. Further, the extracts can be stored desiccated, stable for over a year, and reactivated at the point of use by hydration. The cell free extracts can be programmed by the addition of DNA to allow rapid and simple prototyping of gene circuits for diagnostics or bioproduction.

 In vitro biology provides a number of key advantages for the design, assembly and testing of DNA encoded circuits for diagnostics and environmental sensing. Cell-free extracts avoid the complications, delays and regulatory uncertainty associated with GMOs, while providing opportunities for high level, low cost training and capacity building.

The emerging technology enables engineering of DNA circuits without the need for genetic modification and in a low­ cost manner that makes it accessible for researchers in low­ resource settings. OpenPlant is sponsoring efforts to develop new educational and training materials for use in the UK and developing countries.

Phytobricks: Standard DNA parts for plants (Workpackage B)

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. 

New mechanisms for regulation of gene expression (Workpackage C)

RNA-based mechanisms for gene regulation complement conventional transcriptional regulation, and can be highly flexible and modular. These mechanisms are common in nature, and are only now being harnessed for synthetic systems. OpenPlant aims to expand the availability of genetic regulatory elements across a range of plants, algae and cyanobacteria through projects on riboswitches, riboregulator circuits, cyanobacteria circuits and circadian rhythm regulators.

Molecular tools for targeted genome editing (Workpackage D)

OpenPlant is developing editing tools to enable genome engineering across multiple species and in both nuclear and plastid genomes. We have demonstrated RNA­-guided Cas9-mediated targeted mutagenesis or gene deletion in multiple species including Nicotiana benthamiana, Arabidopsis, tomato, potato, barley, Brassica oleracea, and Marchantia polymorpha. In addition, a standardized toolkit for genome engineering was developed containing a suite of Cas nuclease variants for targeted mutagenesis and gene deletion in multiple plant species (Raitskin et al, 2019).

Digital Tools (Workpackage E)

Software tools play an increasingly important role in Synthetic Biology experiments, as we automate experiments, and as the systems we construct increase in scale. In order to accurately predict the behaviour of biological systems, which are governed by multi-scale, parallel, and feedback-regulated interactions, we need computational models. OpenPlant aims to provide software to (i) automate DNA assembly, (ii) quantify gene expression in plants, and (iii) create models for gene expression and cell growth.