Synthetic Biology brings an engineering approach to biology, broadening the space of applications and promoting new ways of working to facilitate standardization of processes. Synthetic Biology aims to systematically and efficiently iterate through the design-build-test-learn cycle, and rapid and simple prototype systems facilitate this iteration. A simple liverwort, Marchantia polymorpha, has emerged as a test bed for plant synthetic biology and at OpenPlant we have been working on developing tools and resources to exploit the advantages that this system offers.
The first group of resources have been recently published in ACS SynBio (Sauret-Gueto et al, 2020). It includes:
1) Techniques for simple and efficient propagation and maintenance of Marchantia lines throughout its life-cycle, with no requirement for specialized glasshouse facilities.
2) The type IIS OpenPlant DNA toolkit with Loop nuclear transformation vectors, Loop vectors for chloroplast transformation and Loop vectors for CRISPR genome editing. Also included is a collection of standardized DNA L0 parts for expression in Marchantia, including antibiotic resistance genes, signal peptides, fluorescent proteins for multispectral imaging and promoters for gene expression.
3) Techniques for high-throughput imaging of multiple Marchantia lines transformed with different fluorescent markers.
Marchantia is a morphologically simple plant with a dominant haploid phase and a fast life-cycle. It propagates sexually through spores and asexually through gemmae and can be easily maintained in sterile plates and boxes without requirement for glasshouse facilities. Type IIS DNA assembly methods, like Loop, allow for modular, hierarchical and standardized generation of DNA devices, assembling Level (L0) parts into transcriptional units (Level 1, L1) and L1 constructs into Level 2 (L2) devices. The OpenPlant toolkit contains the Loop vectors and a collection of Level 0 parts to use in Marchantia for nuclear and chloroplast transformation and CRISPR genome editing.
In this work, we tested multiple DNA constructs with the idea of producing a collection of optimal DNA parts for Marchantia. Especially important has been our effort to find a promoter expressed in early stages of gemmae development at similar levels in all cells. Also, to develop approaches to understand and engineer morphogenesis, it is important to identify cell-type specific markers. We are currently using the pipeline developed in this work to screen a collection of proximal promoters from the ~400 Marchantia transcription factors, like the new promoter for specific expression in rhizoid cells in Marchantia described in the paper. Other highlights are the Loop vectors for CRISPR and the vectors for chloroplast transformation.
Projected confocal microscopy images showing Marchantia polymorpha gemmae expressing either a fluorescent protein (shown in yellow) targeted to the plasma membrane driven by a constitutive promoter, or three different fluorescent proteins targeted to either the nuclei or the plasma membrane, with one of the fluorescent proteins (showed in green) only accumulating in the rhizoid precursors. From Sauret-Gueto, et al 2020.
The generation of constructs and lines is a team effort in the lab. This effort has been facilitated by tools that enable good data management practices. In particular, we have adopted Benchling because it provides an electronic notebook, molecular biology suite and collaborative data management platform. For more details on how and why we are using Benchling check the recent post in Benchtalk on Generating FAIR — Findable, Accessible, Interoperable, Reusable — Resources with Marchantia, a Prototype for Plant Synthetic Biology.
We have shared the information on the vectors and parts through a public Benchling folder. Also, to ensure that our work is open and accessible, we have made the lab's Marchantia protocols available through the OpenPlant project on protocols.io.
The OpenPlant kit has been well received at the Marchantia workshop in Japan (September 2019) meeting and at the first UK Marchantia meeting in Edinburgh (December 2019). It has already been requested by different labs in UK, Belgium, Switzerland, France, USA, Japan and Mexico. It will also soon be available through Addgene for global distribution under an OpenMTA licence. Use of the OpenMTA places the materials into the public domain, allowing their redistribution and commercial use.
The OpenPlant community is key to the development of all these resources. The OpenPlant toolkit and Marchantia protocols have been mainly developed by former and current researchers in the Haseloff group, at the Department of Plant Sciences, University of Cambridge. But this work has also benefited from interactions among researchers from different groups in OpenPlant, like Nicola Patron group at the Earlham Institute in Norwich, as well as among Researchers with OpenPlant in Cambridge (ROC group), and ROC-Marchantia lovers (specially people from Sebastian Schornack group at SLCU and from Beverly Glover group at the Department of Plant Sciences). There are collaborations going on as a result of these interactions like the ones with the Glover group on CRISPR and the one with Ben Miller group at UEA on extending the collection of tags for Marchantia. Also, many ROC researchers actively participated in discussions on standardisation, tools for data management and sharing.
We would like to thank everyone and are looking forward to more people joining the Marchantia community for basic research and engineering approaches,
Susanna Sauret-Güeto and Eftychios Frangedakis
References:
Sauret-Güeto S, Frangedakis E, Silvestri L, Rebmann M, Tomaselli M, Markel K, Delmans M, West A, Patron NJ, Haseloff J. (2020) Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha. ACS Synth Biol. https://doi.org/10.1021/acssynbio.9b00511