OpenPlant Library of standard DNA parts

Workpackage B: Gene Assembly and Open Registries

As part of the OpenPlant initiative, we are (i) establishing open-source DNA registries in the UK for sharing information, and (ii) exploring new models for distributing plant DNAs and quality control. There is a need to explore multi-tier strategies, as the field is moving quickly from physically defined BioBrick parts, to PCR-defined termini for end-linking, to software-defined parts for direct synthesis. We have established a pilot scheme for open distribution within the OpenPlant project.

OpenPlant embraces state-of-the-art developments in rapid DNA assembly and is building a shared repository of standardised DNA parts across multiple plants, plus algae and cyanobacteria. This will consolidate access to the latest DNA parts for plant synthetic biology, such as improved marker genes, promoters and sensors. Most parts conform to the common syntax for plant synthetic biology. Development of Phytobrick and UNS standards for efficient hierarchical assembly of DNA circuits extends DNA Assembly using Gibson assembly and higher order Mo­Clo and Golden Braid techniques.

PhytoBricks

The PhytoBricks Standard - a common genetic syntax for exchange of DNA parts for plants was introduced in 2015 with wide support from the international plant science community (Patron et al. 2015). Agreement on a standard syntax for parts generated by OpenPlant provides a basis for tackling ongoing work on automated DNA assembly and the establishment of a central registry for plant parts. 

In addition, the inclusion of the standard as BioBricks Foundation RFC 106 as a collaborative output from the Cambridge, Valencia and Norwich Research Park iGEM teams led to i) acceptance of PhytoBricks parts in iGEM ii) the establishment of an inaugural Plant Prize at the 2016 iGEM competition iii) access to the iGEM Registry as a low cost, global method for description and distribution of PhytoBrick DNAs.

Details of the PhytoBrick standard and comprehensive instructions for how to synthesize and clone parts for distribution can now be found on the iGEM website.

Type IIS restriction endonuclease mediated cloning

• Type IIS-mediated assembly methods are based on the original description of "Golden Gate" cloning and allows efficient simultaneous assembly of multiple DNA parts, without requiring the isolation of individual fragments.

Nicola Patron has assembled a short online course on Type IIS that covers:

• How type IIS restriction enzymes enable a one-pot reaction

• How to easily and reliably assemble multiple standard parts in a single reaction, and

• How to make new parts

You can order the Universal Acceptor Plasmid, pUAP1 and other plasmids via Addgene.

Automation

OpenPlant aims to produce a fully automated workflow for the design and assembly of DNA constructions. We are also introducing automation into phenotyping and analysis workflows.

Liquid Handling

Labcyte Echo acoustic focusing liquid handling robots for automating DNA assembly have been installed in the Haseloff Lab and the Earlham Institute. Shared protocols are being developed for a range of lab tasks.

A Gilson PIPETMAX robot has also been installed in the Haseloff Lab for use with the open source Antha language.

Similar integrated procedures for DNA assembly and storage have been developed at JBEI, Synthetic Genomics, Biofab and Ginkgo Bioworks.

Credit: Labcyte Echo

Software automation for DNA assembly

The Gibthon Construct Designer is a Cambridge-designed web service which allows one to automatically generate primers for Gibson Assembly. The software arose from an iGEM team project, and it provides sequence design tools and automates the design of the oligos for rapid assembly of DNAs. 

Collections

MarpoDB

MarpoDB is a gene-centric resource of Marchantia polymorpha Cam-1 genetic parts for engineering and plant synthetic biology.

Addgene

PhytoBricks compliant plant parts are already being shared through the Addgene and a wider collection of parts from algae and cyanobacteria will also be added as OpenPlant progresses.

Phytobricks in the iGEM registry

The iGEM Registry now contains a number of plant PhytoBricks and assembly vectors in addition to a collection of plant parts from previous iGEM teams.

Material Transfer Agreement

OpenPlant is 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. Read more about the OpenMTA.

Publications

Srivastava V, Amanna R, Rowden SJL, Sengupta S, Madhu S, Howe CJ, Wangikar PP (2021) Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance. Journal of Bioscience and Bioengineering. doi:10.1016/j.jbiosc.2020.11.012 PMID: 33610455

Grant PK, Szep G, Patange O, Halatek J, Coppard V, Csikász-Nagy A, Haseloff J, Locke JCW, Dalchau N & Phillips A. (2020). Interpretation of morphogen gradients by a synthetic bistable circuit. Nature Communications 11: 5545 https://doi.org/10.1038/s41467-020-19098-w

Dorrell R, Nisbet E, Barbrook A, Rowden S, Howe C. (2019) Integrated genomic and transcriptomic analysis of the peridinin dinoflagellate Amphidinium carterae plastid. Protist 170(4) 358-373 https://doi.org/10.1016/j.protis.2019.06.001

Wey LT, Bombelli P, Chen X, Lawrence JM, Rabideau CM, Rowden SJL, Zhang JZ & Howe CJ. (2019) The Development of Biophotovoltaic Systems for Power Generation and Biological Analysis. ChemElectroChem. 6(21):5375-5386. doi: 10.1002/celc.201900997.

Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, Lea-Smith DJ, McCormick AJ. CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax. Plant Physiol. 2019 May;180(1):39-55. doi: 10.1104/pp.18.01401. Epub 2019 Feb 28.

Martins BMC, Tooke AK, Thomas P, Locke JCW, (2018). Cell size control driven by the circadian clock and environment in cyanobacteria. Proc Natl Acad Sci U S A. 115(48):E11415-E11424. doi: 10.1073/pnas.1811309115.

Pollak B, Cerda A, Delmans M, Álamos S, Moyano T, West A, Gutiérrez RA, Patron NJ, Federici F, Haseloff J (2018) Loop Assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol. 222(1):628-640. doi: 10.1111/nph.15625.

Kahl L, Molloy J, Patron N, Matthewman C, Haseloff J, Grewal D, Johnson R, Endy D. (2018) Opening options for material transfer. Nat Biotechnol. 36(10):923-927. doi: 10.1038/nbt.4263.

Vazquez-Vilar M, Orzaez D, and Patron, N (2018) DNA Assembly Standards: Setting the Low-Level Programing Code for Plant Biotechnology. Plant Sci. 273:33-41. doi: 10.1016/j.plantsci.2018.02.024.

Rowden S, Bombelli P and Howe C (2018) Design and study of bio-electrochemical system for biotechnological applications and metabolic investigation in Photosynthesis. Methods Mol Biol. 1770:335-346. doi: 10.1007/978-1-4939-7786-4_20.

Bateson P, Fleet JEH, Riseley AS, Janeva E, Marcella AS, Farinea C, Kuptsova M, Conde Pueyo N, Howe CJ, Bombelli P, Parker BM (2018). Electrochemical Characterisation of Bio-Bottle-Voltaic (BBV) Systems Operated with Algae and Built with Recycled Materials. Biology (Basel) 7(2). pii: E26. doi: 10.3390/biology7020026.

Delmans M, Pollak B, and Haseloff J, (2017). MarpoDB: An open registry for Marchantia polymorpha genetic parts. Plant Cell Physiol. 58(1):e5. doi: 10.1093/pcp/pcw201.

Martins BM, Das AK, Antunes L, Locke JC (2016). Frequency doubling in the cyanobacterial circadian clock. Mol Syst Biol. 12(12):896. doi: 10.15252/msb.20167087.

Patron N. (2016). DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:604-616 https://doi.org/10.1002/cppb.20038

Patron NJ (2016) Synthetic Plants. Synthetic Biology Handbook. Ed: Darren Nesbeth. CRC Press. ISBN 978146656847

Patron NJ (2016). Blueprints for Green Biotechnology: Development and Application of Standards for Plant Synthetic Biology. Biochem Soc Trans. 44(3):702-8. doi: 10.1042/BST20160044.

Patron NJ (2016). Synthetic Biology and Gene Cloning. Encyclopedia of Applied Plant Sciences 2nd Edition. Editor-in-Chiefs: Brian Thomas Denis and J Murphy Brian G Murray. Academic Press. eBook ISBN: 9780123948083 Hardcover ISBN: 9780123948076

Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith A, Hibberd J, Webb AA, Locke J, Schornack S, Ajioka J, Baulcombe DC, Zipfel C, Kamoun S, Jones JD, Kuhn H, Robatzek S, Van Esse HP, Sanders D, Oldroyd G, Martin C, Field R, O'Connor S, Fox S, Wulff B, Miller B, Breakspear A, Radhakrishnan G, Delaux PM, Loqué D, Granell A, Tissier A, Shih P, Brutnell TP, Quick WP, Rischer H, Fraser PD, Aharoni A, Raines C, South PF, Ané JM, Hamberger BR, Langdale J, Stougaard J, Bouwmeester H, Udvardi M, Murray JA, Ntoukakis V, Schäfer P, Denby K, Edwards KJ, Osbourn A, Haseloff J. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist 208(1):13-9. doi: 10.1111/nph.13532.

Rutten V, Munabi A, Riche F, Lewy G, Wilson H, Pipan M, Bhate S, Nghiem T-A, Kaufhold W, Haseloff J, Rubert A, González A, Quijano A, Llopis I, Gavaldá J, Estellés L, Vásquez M, Orzáez D, Deal C, Gray J, Spiegel M, Monsey S, Middlemiss A, Day J, Patron NJ (2015) A Standard Type IIS Syntax for Plants. RFC #106 (http://hdl.handle.net/1721.1/96069)