OpenPlant

Lead Research Organisation: University of Cambridge
Department Name: Plant Sciences

Abstract

An essential concept in Synthetic Biology is that genetic systems can be constructed using standardised, interchangeable parts. For example, functional DNA sequences such as promoters, ribosome binding sites and coding sequences can be used to develop synthetic gene systems that can be used to reprogram living systems in a systematic way. The prospect of large-scale reprogramming of living systems, will require access to relatively large numbers of components. This is in contrast to existing GM products that contain one or a few components. There is growing requirement for libraries of well characterized routine components that can be shared for the construction of a variety of systems, where small companies would be ensured freedom to operate. This is essential to foster the kind of innovation seen at the emergence of other new technologies such as microelectronics and software development.

Accordingly, a substantial part of the Synthetic Biology field has promoted open standards and sharing of data and resources, inspired by the open source software movement. The educational community has embraced the open source principle, and DNA parts are widely and freely distributed internationally as part of educational efforts like the iGEM community, and grows year-on-year. The BioBricks Foundation is a non-profit organisation which has roots in the academic community, and which has promoted a legal framework which would allow protection of Synthetic Biology applications and key activities, but which would facilitate sharing of parts.

Plant biotechnology is a good example of a field where current IP practices have led to a restriction of exchange and innovation, and this model is threatening to predominate in plant synthetic biology. For examples, one just has to look at restrictive licensing of basic technologies like transformation, selection markers and useful genes. 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. As the speed and scale of biological assembly increases, this becomes more pressing.

As part of the OpenPlant initiative, we will establish open-source DNA registries across the UK for sharing information, and to join an international web of registries with plant specific parts. The technology is inherently low cost, renewable and has obvious applications for new sustainable technologies.

Technical Summary

Foundational technologies: As part of the OpenPlant initiative, we will (i) establish open-source DNA registries for sharing information, and join an international web of registries with the first plant specific parts. (ii) We will develop a major new plant chassis for Synthetic Biology, (iii) develop new DNA parts for the control and quantitative imaging of genetic circuits in plants, and (iv) genome-scale engineering in plants. (v) Software tools will be further improved for automated DNA assembly, modeling of synthetic gene circuits and cellular morphogenesis

Trait Engineering: The development of new foundational tools and parts will directly contribute to the engineering of new traits in plants, such as (i) altered photosynthesis and leaf structure, (ii) carbohydrate content, (iii) metabolic pathways, (iv) new forms of symbiosis and nitrogen fixation and (v) high level production of biomolecules by virus engines.

Open technologies for innovation: 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 will create new forums and opportunities for open innovation in plant synthetic biology.

We will sponsor discussions in Cambridge on the potential impact of Synthetic Biology on sustainable practices in agriculture, bioproduction, land use and environmental conservation. This will bring together a wide range of engineers, scientists and policy developers to explore new technologies and possible models for sustainable agriculture, bioproduction and land use.

Planned Impact

A significant barrier to the ability of Synthetic Biology to have commercial impact in the UK is social acceptance of genetic modification. OpenPlant will engage with this issue scientifically by the development of precision gene technologies that can introduce desirable traits without the introduction of transgenes and by supporting research on traits such as nitrogen use that can significantly impact bioproduction. It will also engage through discourse on sustainable agriculture and land use and engagement with a variety of groups including conservationists, social scientists and policy developers.

The restrictive licensing of powerful technologies as well as the prohibitive cost of protecting IP for smaller enterprises threatens to impede the pace of research in plant synthetic biology. The OpenPlant centre aims to promote entrepreneurial activities though a framework that allows the exploration of open technologies and fixed-cost, non-exclusive licenses that can ensure that parts, techniques, libraries and materials have are available to facilitate both scientific exchange and commercial innovation.

Publications

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Saxena P (2016) Virus-Derived Vectors for the Expression of Multiple Proteins in Plants. in Methods in molecular biology (Clifton, N.J.)

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Hibberd JM (2016) Wheat genomics: Seeds of C photosynthesis. in Nature plants

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Patron N (2016) Synthetic Biology Handbook

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Boutanaev AM (2015) Investigation of terpene diversification across multiple sequenced plant genomes. in Proceedings of the National Academy of Sciences of the United States of America

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Mestecky Jiri (2015) Mucosal Immunology

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N├╝tzmann HW (2015) Regulation of metabolic gene clusters in Arabidopsis thaliana. in The New phytologist

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Scaife MA (2015) Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. in The Plant journal : for cell and molecular biology

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Medema MH (2015) Minimum Information about a Biosynthetic Gene cluster. in Nature chemical biology

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Delaux PM (2015) Algal ancestor of land plants was preadapted for symbiosis. in Proceedings of the National Academy of Sciences of the United States of America

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Belhaj K (2015) Editing plant genomes with CRISPR/Cas9 in Current Opinion in Biotechnology

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Delaux PM (2015) Tracing the evolutionary path to nitrogen-fixing crops. in Current opinion in plant biology

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Thimmappa R (2014) Triterpene biosynthesis in plants. in Annual review of plant biology

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Sainsbury F (2014) Transient expressions of synthetic biology in plants. in Current opinion in plant biology

 
Title Anxiety and Awe, a collection of poems by Norwich Scientists and Writers 
Description The 2015 NRP-UEA iGEM team worked with the Science and Writing Trust (SAW), to hold a workshop for established poets at the amazing Dragon Hall, a beautiful medieval building and home to the Norwich Writers Centre. At the workshop, we talked about our project, iGEM and bioengineering with established and published poets. We conducted writing exercises guided by the published poet, Esther Morgan in which we expressed our emotions, concerns and awe of science. This workshop resulted in a collaborative collection of poems by writers and scientists. The collection is published on our website (http://2015.igem.org/Team:NRP-UEA-Norwich/WritersWorkshop) and was presented at the iGEM Giant Jamboree, where it gained us a nomination for best communication. 
Type Of Art Creative Writing 
Year Produced 2015 
Impact The collection is published on our website (http://2015.igem.org/Team:NRP-UEA-Norwich/WritersWorkshop) and was presented at the iGEM Giant Jamboree, where it gained us a nomination for best communication. 
URL http://2015.igem.org/Team:NRP-UEA-Norwich/WritersWorkshop
 
Title Glowing oat seedlings 
Description Young oat seedling roots glow under UV due to the accumulation of the antimicrobial triterpene avenacins. Oats naturally produce avenacins exclusively within the root tip epidermal cells, which protects them from soil pathogens. Image supplied by researchers in the Osbourn laboratory at the John Innes Centre on the Norwich Research Park. 
Type Of Art Image 
Year Produced 2016 
Impact Image placed on the Norwich Research Park image library, for free sharing and dissemination. 
URL http://images.norwichresearchpark.ac.uk/imagedetails.aspx?imgid=229
 
Title Image of Nicotiana benthamiana 
Description Nicotiana benthamiana, a relative of the tobacco plant, is commonly used in plant research. Here, the leaf is used as a host organism to produce components of avenacin, a fluorescent antimicrobial produced by oat roots, which protects the roots from soil pathogens. A cluster of genes - the Sad genes - have been identified in oat as the instructions used to produce avenacin. The blue spots indicate the presence of parts of the avenacin compound introduced by inserting some of the Sad genes into the host plant. The other spots are controls to test the expression system. Image supplied by Aymeric Leveau, Osbourn laboratory. 
Type Of Art Image 
Year Produced 2015 
Impact Image placed on the Norwich Research Park Image Library, for free sharing and dissemination. 
URL http://images.norwichresearchpark.ac.uk/imagedetails.aspx?imgid=103
 
Title Nicotiana, biofoundry. 
Description An artistic interpretation inspired by plant systems for bioproduction of natural products developed with artist and designer, Karen Ingram. 
Type Of Art Artwork 
Year Produced 2018 
Impact Published as part of 'Convergent Visions' Faesthetic #15 produced in partnership with SXSW for the SXSW Art Program 2018. Presented at SXSW Interactive 2018. 
URL http://think.faesthetic.com/archives/9687
 
Title OpenMTA Video 
Description A video describing the open material transfer agreement produced by the Biobricks Foundation in collaboration and featuring interviews with OpenPlant group leaders and others. 
Type Of Art Film/Video/Animation 
Year Produced 2016 
Impact The OpenMTA has not yet been officially launched so there are so far only 71 views on youtube. 
URL https://www.youtube.com/watch?v=zt6b8_mY-i4
 
Description Key Findings


2014-2015

Refurbishment and equipping of new OpenPlant laboratories
in Cambridge and Norwich.

Establishment of a common genetic syntax for exchange
of DNA parts for plants, extensible to all eukaryotes (Patron et al., 2015; RFC106).

Drafting of an Open Materials Transfer Agreement, a
simple, standardized legal tool to enable sharing of materials and associated data on a more open basis.

Implementation of a "single-click" OSX-installable version
of the JBEIR-ICE open source DNA registry and DNA manipulation software.

Development of routine methods for transformation and
gene editing in Marchantia polymorpha.

Development of Marchantia paleacea as a new system for
engineering actinomycorrhizal associations.

Generation of draft genome and transcriptome maps for
M. polymorpha and M. paleacea.

Characterisation of miR1157 and miR1162 precursors for
use as synthetic gene regulators in Chlamydomonas reinhardtii.

Refactoring and use of the HyperTrans system for rapid
testing of DNA circuits for terpene synthesis in Nicotiana benthamiana.


2015-2016

Commissioned advanced imaging/robotics equipment at
the Cambridge OpenPlant lab.

Completion of genome sequence and transcript map of
the Cam-1 (male) and Cam-2 (female) isolates of Marchantia polymorpha. Data will be included in forthcoming publication of genome.

High resolution map of the time course of gene expression
during sporeling germination and chloroplast differentiation.

Construction of MarpoDB, a genecentric database for
mining and describing DNA parts from Marchantia (http://marpodb.io)

Official acceptance of the common syntax for plant DNA
parts as a new standard (Phytobricks) in the iGEM 2016 competition, and introduction of an award for plant work.

Development of Phytobrick and UNS standards for efficient
hierarchical assembly of DNA circuits.

Expansion of a Chlamydomonas DNA toolkit for target
gene expression and assay of miRNA-dependent gene silencing.

Development of a suite of Cas9 variants and toolkit
for targeted mutagenesis and gene deletion in multiple plant species.

Construction of series of vectors for fine tuning of
protein expression using HyperTrans system

Methods for mining plant genomes for biosynthetic gene
clusters

Design and synthesis of an artificial protein scaffold
library, built to the Phytobrick standard and verified by BiFC.

Production of cell-specific epitope tags for identifying
DNA motifs that drive gene expression in photosynthetic tissues in Arabidopsis.

Publication of a novel reporter for chloroplast transformation,
and identification of transit peptides for chloroplast localisation of nuclear encoded products in Marchantia.

Identification of a large repertoire of carbohydrate
active enzymes in Euglena gracilis.

Transformation of gene editing constructs into potato,
to create digestion-resistant starches, and preliminary screening of transformed plantlets.

Gram-scale production of triterpenes for analysis and
assay, using the HyperTrans system.

Production of the plant-derived iridoid alkaloid strictosidine
in yeast.

Generation of a trichome-specific protein database for
enzyme discovery.

Asteraceae P450 proteins as a toolkit for targeted modification
of sesquiterpenes.

Development of the HyperTrans system for use in tomato.

Screening tomato introgression lines for regulators
of monoterpene biosynthesis.

Yeast one-hybrid analysis for the identification of
transcription factors that regulate triterpene metabolic gene clusters.

Characterisation of gene targets for AtMYB12 and SIMYB12
in tomato, for enhancing phenylpropanoid metabolism and high levels of resveratrol and genistin production.

Construction of a synthetic gene cluster for dhurrin
biosynthesis in Arabidopsis roots.

Construction and distribution of HyperTrans DNA vectors
that are compatible with the Phytobrick standard.

Testing of the HyperTrans system in Marchantia and BY2
cells.

Consultation on the design of the Norwich Research Park
LeafSystems high throughput production facility, due for completion in Q2 2017.


2016-2017

Consolidation of the Phytobrick standard for Type IIS
based DNA parts for plants, including acceptance as first standard for eukaryotic DNA parts, and introduction of the Plant Prize in iGEM 2016.

Design, domestication and synthesis of the first 500
DNA parts for Marchantia (Susana Sauret-Gueto, Haseloff lab).

Establishment of the Loop assembly technique first presented
at the iGEM2016 Jamboree (Bernardo Pollak & Fernan Federici, Haseloff lab).

Automation of Loop assembly at 500nL scale using Labcyte
acoustic focusing and Hamilton robots at Earlham Institute (Patron lab, A. Hall lab & Haseloff lab).

Established an automated assembly and transient expression
workflow for assessing Cas9-mediated targeted mutagenesis using plant protoplasts and Illumina MiSeq sequencing (Oleg Raitskin, Patron Lab).

Validated an expanded molecular toolkit in the plant
common syntax of Cpf1- and Cas9- variants showing improved specificity in plant protoplasts (Raitskin, Patron Lab).

Established methods for direct delivery of a ribonuclease
complex (Cas9:YFP recombinant protein & guide RNA) for induction of targeted mutagenesis in protoplasts of N. tabacum, N. benthamiana & S. tuberosum (Raitskin, Patron Lab).

Developed S. elongatus constructs for examining the
circadian clock and its outputs at the single cell level, and published frequency doubling model of the clock in Molecular Systems Biology (Bruno Martins, Locke lab).

Circadian clock model made available in SBML format
(Martins, Locke lab).

Validated the use of fluorescent protein based reporter
for evaluating miRNA mediated gene silencing in Chlamydomonas (Francisco Navarro, Baulcombe lab).

Optimised miRNA abundance, extent of sequence complementarity
and target sites for gene silencing in Chlamydomonas (Navarro, Baulcombe lab).

Constructing synthetic genetic circuits with miRNA mediated
incoherent feed-forward loop to confer robust levels of gene expression (Navarro, Baulcombe lab).

Constructing DNA parts, in accordance with the common
syntax, to contribute to a Chlamydomonas MoClo toolkit (Baulcombe and Smith labs).

Synthesis of a panel of codon optimised fluorescent
reporters spanning the visual spectrum, including five variants of the fluorescent reporters: iRFP670, mCardinal, mPlum, mCerulean, mNeptune, mRaspberry, mTurquoise, mWasabi, eBFP, Sirius and TagCFP, all modified for chloroplast expression (Ajioka lab).

The Marchantia chloroplast genome has been re-annotated.
Missing ORFs have been identified, partial annotations have been completed and likely promoter sites identified throughout the chloroplast genome using BPROM (Ajioka lab).

CRISPR-Cas9 mediated gene KO to produce "giant chloroplast"
phenotypes in Marchantia, (Male, Pollak, Sauret-Gueto, Silvestri in Haseloff lab).

Established reproducible colonisation of several liverwort
species (Marchantia spp., Lunularia cruciata) with Glomeromycota fungi (Funnelliformis mossae, Rhizophagus irregularis) in custom vermiculite system and detection using staining and high resolution confocal fluorescence microscopy (Philip Carella, Schornack
lab).

Established constructs for secretion system pathway
and tonoplast labelling and have confirmed functionality in M. polymorpha (Carella, Schornack lab).

Enhancer trap screen underway in Haseloff lab (Tomaselli,
Sauret-Gueto & Silvestri.

In planta cytometry techniques developed in Marchantia
gemma (Pollak & Delmans, Haseloff lab).

Developed clearing techniques for image reconstruction
of Marchantia air chambers (Marta Tomaselli, Haseloff lab) .

Built ubiquitin-tagged rapid-turnover fluorescent markers
for imaging dynamic genetic responses (Lukas Mueller, Webb-Haseloff labs).

New synthetic version of the 5' UTR used in HyperTrans
system has been shown to be twice as effective as the original HT sequence (Hadrian Peyret, Lomonossoff lab).

Developed a new vector system (pEFF) which combines
the high translational benefits of the CPMV-HT system with the replication ability of potato virus X, in collaboration with the Centre for Bioengineering at the Russian Academy of Sciences (Lomonossoff lab).

Characterised promoter elements that drive specific
expression in leaves, and a negative regulator that represses expression in mesophyll and veinal cells (Hibberd lab).

Compiled a list of transcription factors preferentially
expressed in bundle sheath cells of Arabidopsis, including cognate TF for above-mentioned promoter element (Hibberd lab).

Heterologous xylan arabinosyltransferases have been
cloned in Arabidopsis lines using common syntax rules (Henry Temple & Louis Wilson, Dupree lab).

Testing the enzymatic activities of arabinosyltransferases
using N. benthamiana transient expression with the HyperTrans vector system (Temple & Wilson, Dupree lab).

Exploring artificial in vitro metabolic cycles driving
production of glucose-based oligosaccharides from cheap and readily available sucrose by using sucrose phosphorylase and glucan phosphorylases (Field lab).

Applying and testing the genome editing tools and technologies
developed in the Patron lab to generate potatoes that contain digestion-resistant starches with potential nutritional benefits (Aytug Tuncel, Smith lab, JIC).

Developed methods to transform and engineer protoplasts
directly isolated from potato leaves (Tuncel & Raitskin, Smith and Patron labs).

Mined transcriptome of Euglena gracilis for metabolic
carbohydrate metabolism and natural product biochemistry and released in the CAZy database (Field lab).

Collaborated with Medema lab (Wageningen) to release
plantiSMASH, a customised algorithm for mining plant genomes for biosynthetic gene clusters (Kautsar et al. 2017; Osbourn lab).

Improved agro-infiltration methodology for production
of triterpenes using the HyperTrans transient plant expression system, leading to gram-scale production of the triterpene scaffold, ß-amyrin (Reed et al., 2017; Osbourn lab).

Identified genes that modulate stereochemistry of the
iridoid ring system, with potential for compounds with important agrichemical activity (Benjamin Lichman, O'Connor lab.

Blocking turnover of L-DOPA production in beetroot to
enable low-tech plant sourced bioproduction for Parkinson's treatment (Noam Chayut, Martin lab)

Engineered enzymes from the Asteraceae family into yeast
to generate oxygenated sesquiterpenes (Nguyen et al., 2016; Don Nguyen, ex O'Maille lab).

Investigating three-dimensional positioning of biosynthetic
gene clusters in the A. thaliana genome using Hi-C mapping and FISH analysis (Hans Nützmann, Osbourn lab)

Yeast-one-hybrid assays have been used to identify a
candidate transcription factor for a biosynthetic gene cluster from oat (the avenacin cluster; Osbourn lab).

Nine promoters have been isolated from the oat avenacin
cluster that retain root-specific expression patterns in a wide range of plants (Aymeric Leveau, Osbourn lab).

Three of these oat promoters have been used to drive
the expression of a 3 gene pathway for a plant defence compound (dhurrin) from sorghum in Arabidopsis roots (Osbourn lab).

Produced synthetic virus-like particles (VLPs) to enable
the structure of particles of potato leafroll virus to be solved to near atomic resolution (Lomonossoff lab).


2017-2018

Publication of quantitative microscale work showing
intercellular adhesion promotes clonal mixing in growing bacterial populations.

Development of microfluidic vessels for quantitative
observation of cyanobacteria.

Droplet-based microfluidic analysis and screening of
single plant cells.

Construction of an improved generation of Loop Assembly-compatible
transformation vectors for plants.

Establishment of efficient chloroplast transformation
techniques in Marchantia.

Use of multispectral markers with in planta cytometry,
to allow segmentation of cell geometries and quantitative assignment of biological parameters on a cell-by-cell basis.

Publication of the genome sequence and transcriptome
datasets for Marchantia.

Publication of revision 4.0 of MarpoDB

Synthesis of an almost complete collection (350/398)
of proximal promoters for transcription factors in Marchantia.

Collection of single-cell transcriptomic data from germinating
gemma.

Launch of the OpenMTA license with a published commentary
in Nature Biotechnology 36:923-927, 2018.

Screening of cell and tissue type specific expression
lines from enhancer trap transformants.

Development of a novel system for "resetting" Marchantia
development by surgically dissecting gemma.

Miniaturisation and automation of efficient DNA Loop
Assembly reactions using nanolitre scale handling.

Integration of Benchling as a free web-based solution
for online lab notebooks, sequence editor and inventory handler.

Use of Benchling as a DNA registry and for protocol
sharing across OpenPlant.

Construction of optimised genetic circuits as reporters
for circadian clock output.

Identification of a number of putative electrically
regulated promoters in cyanobacteria.

Characterisation of improved biophotovoltaic vessels
and chassis strains.

Construction and publication of a MoClo kit, consisting
in a set of Golden Gate-domesticated DNA parts, for the green alga Chlamydomonas reinhardtii.

High-throughput workflow to analyse conditional behavior
of riboswitches in vivo.

MoClo parts for transformation of the chloroplast genome
of C. reinhardtii.

Establishment of protocols to allow chloroplast transformation
by electroporation in Chlamydomonas.

Regulation of chloroplast gene expression via TPR/PPR
proteins encoded by the nucleus in Chlamydomonas.

Sequencing and reannotation of the chloroplast genome
from the Cambridge isolates of M. polymorpha.

Construction of a new generation of Loop Assembly vectors
for plastid transformation in Marchantia.

Optimisation of biolistic delivery protocols for high
efficiency plastid transformation in Marchantia.

Generation of ftsz1 and ftsz2 mutant plant lines that
produce fewer and larger plastids per cell.

Development of techniques and novel vector systems for
RNA-guided Cas9-mediated targeted mutagenesis and gene deletion in plant species (Nicotiana, Arabidopsis, Marchantia).

Automated construct assembly and delivery to plant protoplasts
to enable rapid quantitative assessments of the efficiency and specificity of gene editing.

Development of software-based 3-parameter measurement
techniques for quantifying gene expression in cell suspensions to minimise extrinsic noise

Publication of a computational framework for automated
analysis of microplate reader data.

Construction of models for the coupling of the cyanobacterial
clock and control of period and cell growth.

Development of software-based classification schemes
for description of gene expression at the cellular scale in Marchantia gemmae.

Maintenance and upgrades for the CellModeller software
package, to include cell-cell adhesion and cell shape.

Publication of algorithms for whole colony-scale segmentation
from confocal microscopy datasets of growing microbes.

Identification and functional characterisation of DNA
parts for natural product synthesis.

Employment of genomic neighbourhood associations to
identify triterpene-scaffolding genes with tailoring cytochrome P450s and acyltransferases.

Improvement of agro-infiltration methodology for gram-scale
production of triterpenes using the HyperTrans transient plant expression system.

Publication of the improved infiltration protocol in
text and video format.

In silico identification of new CYP450s with different
oxidizing specificities.

Identification of new building blocks for biosynthesis
of iridoids.

Discovery of neofunctionalisation of a short chain alcohol
dehydrogenase (SDR) with a non-redox role in controlling the stereochemical course of ring cyclization during iridoid synthesis.

Blocked the turnover of L-DOPA in beetroot, using CRISPR/Cas9-mediated
genome editing, to enable low-tech accessible production in a plant system.

Development of a novel suspension culture production
system that produces exceptionally high levels of anthocyanins.

Over-expression of the Rosea1 and Delila transcription
factors to produce multiply-acylated blue anthocyanins.

Activation of the anthocyanin biosynthetic pathway in
cultures of Arabidopsis thaliana to produce diacylated cyanidin with a blue colour at neutral pH.

Identification and characterisation of transcription
factors for control of natural product production.

Use of protocols for chromosome conformation capture
and FISH analysis to investigate the three-dimensional positioning of biosynthetic gene clusters in the nucleus of Arabidopsis thaliana.

Identification of candidate transcription factors that
regulate promoters of metabolic gene clusters including central pathway cluste