CyanoSource: A foundry generated barcoded mutant library resource for the model cyanobacterium Synechocystis sp. PCC 6803

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

Cyanobacteria are an evolutionarily ancient and abundant group of oxygenic photosynthetic bacteria that together account for ~25% of global carbon fixation. Several species are used for food, nutraceuticals and dye production, in an industry currently worth >£500m. Others are potential platforms for synthesis of pharmaceuticals, industrial chemicals and biofuels. They are particularly suited for the production of plant drugs, a market worth over £20b in the last decade, since many physiological and biochemical features are conserved between cyanobacteria and plants. Despite their importance, our knowledge of many key aspects of cyanobacterial biology is limited, which impedes fundamental understanding and the development of cyanobacteria as efficient biotechnology platforms.

A key technique used to understand the function of genes is the generation and characterisation of mutants in which a specific gene, or genes of interest, has been deleted. However, generation of cyanobacterial mutants is time consuming, costly and requires specific expertise that is not available to all research groups. Moreover, research groups use different sub-strains that may differ at the genotype or phenotype level. This leads to issues with reproducibility of results between different studies, which is increasingly recognised as a problem in scientific research. Furthermore, failure to publish results can lead to labs generating (or attempting to generate) the same mutants, resulting in unnecessary replication of experiments and resource wastage.

In this project we will use two recently established UK DNA foundries located in Edinburgh and Norwich, respectively, which contain automated robotics technologies that are able to rapidly construct a whole genome mutant library. Using the foundries, we will target 3,456 genes in the model cyanobacterium, Synechocystis sp. PCC 6803 (Synechocystis). Synechocystis is the most widely studied cyanobacterium and has a number of advantages for biotechnology. It can be genetically manipulated, grows rapidly when bubbled with carbon dioxide, and tolerates a wide range of environmental conditions. To generate mutants, we will automate the assembly of gene knockout plasmids that will be introduced into Synechocystis, which naturally imports DNA. Synechocystis will be transformed via a robust process called homologous recombination, which will result in a complete library of knockout mutants. This resource will greatly assist the research community in gene function studies.

Generation of this mutant library will also allow us to determine which genes are essential for survival in Synechocystis under typical laboratory growth conditions. Conditional mutants (i.e. specialised mutants that require an external stimulus to repress a gene) will be constructed for essential genes that cannot be removed. Here, we will use a copper sensitive promoter that switches off the gene when copper is present. All plasmids and mutants will be made available to UK and international researchers via a public database, which will be updated throughout the project. Overall, these resources will significantly advance cyanobacterial research and the development of strains for biotechnology applications.

Technical Summary

Our goal is to develop a much needed resource for cyanobacterial research to assist with the systematic analyses of unknown gene functions and gene regulatory networks and also for genome-wide testing of mutational effects in a common sub-strain of the model Synechocystis sp. PCC 6803 (Synechocystis). Building on the transformation and Modular Cloning (MoClo) techniques pioneered by the applicants, we will collaborate with UK DNA foundries in Norwich and Edinburgh to generate a whole genome library of gene insertion plasmids (representing 3,456 coding sequences) and produce the largest available collection of known and novel cyanobacterial mutant strains in the world.

The CyanoSource collection will contain a library of barcoded mutants for all non-essential genes that can be used for individual gene functional studies, or analysed as a pool to investigate the roles of individual genes in a given growth environment. Secondly, it will also include a unique set of conditional mutants targeting genes that are essential for survival under typical growth conditions. In the latter collection, the promoters of essential genes will be replaced seamlessly with the copper repressible promoter of the petJ gene. Thirdly, CyanoSource will make available the library of plasmids used to generate the mutants. The gene knockout plasmids will be designed with MoClo compatible cloning sites to facilitate additional targeted genome engineering applications. Lastly we will generate a publicly available dataset containing information on available plasmids and mutants that will be linked with two bioinformatic tools recently developed by the applicants, a sub-cellular proteome location map and a comparative genomics dataset, to form a single, easily accessible platform. Users will be able to identify highly conserved cyanobacterial genes, their subcellular location within Synechocystis, and order mutants and plasmids of interest.

Planned Impact

Both PIs will oversee and co-ordinate impact activities. DLS and AM have excellent experience of involvement in projects with impact (see Track Records).

Who will benefit from this resource and how?
This project provides an outstanding opportunity to integrate the skillsets of two cyanobacterial researchers with the DNA foundries at their respective institutions. Both PIs have been successful in investigating a broad range of questions related to cyanobacteria. This resource will rapidly advance their work, which will be of benefit to other researchers working in the field. This project will facilitate exchange between the foundries in Norwich and Edinburgh, leading to improvements in their bioinformatics and automation techniques. During the development of CyanoSource, the DNA foundries will also gain the opportunity to develop collaborations with other academics and industries. The PDRAs and students working on the project will benefit considerably from training at DNA foundry facilities. Thus, this project will help to train the next generation of researchers in microbiology, synthetic biology and automation. The general public will benefit from planned outreach activities. Furthermore, the availability of this resource will accelerate our understanding of the fundamental biology in oxygenic phototrophic prokaryotes and thus have wide educational value at all levels, through schools and universities.

In the short to medium term, the outputs of CyanoSource will benefit academics and researchers performing fundamental cyanobacterial research in the UK. Particularly, this work will be of interest to researchers focused on gene function studies. It will also be of considerable interest to metabolic engineers and metabolic modellers. In the medium to long term, the beneficiaries will include international academic researchers and industrial researchers. Both PIs have connections to UK companies working in the biotechnology sector (i.e. ScotBio, Cyanetics). Mutants and plasmids will be available to order for companies at similar cost to other users, while researchers in lower middle income countries will be able to obtain mutants free of charge (although they will have to cover transport costs).

How will we ensure they benefit from the resource?
Both PIs are members of Algae-UK, a Phase II BBSRC NIBB consortium of academics and industry committed to developing a UK based cyanobacterial/algal biotechnology industry. All relevant information from this project will be disseminated at Algae-UK meetings and international conferences. We will maintain an up-to-date website for CyanoSource and release a monthly newsletter (through Algae-UK) to publicise the latest plasmids and mutants produced. We will publish results in high-impact journals in a timely fashion with open access. Furthermore, we will work with our external relations teams to promote our research to a broad spectrum of end-users via press releases, contacts with journalists, and specialised news media. The use of social media tools (e.g. YouTube) will also provide a contemporary forum for knowledge exchange (see PtI). We will make use of existing contacts with PIs (see list of LoS), industry and other academics with particular research interests as soon as relevant materials are generated at CyanoSource. We will recognise and protect user confidentiality for mutants ordered, to ensure no conflicts of interest arise. We will provide mentoring to ensure uptake of PDRA training schemes, including regular progress reviews and career development plans, and participation in the promotion of CyanoSource. Results will be used as part of our regular engagement with students and non-academic audiences through outreach activities.
 
Description 2020 March. Since the start of the award (Sept 2019), we have produced one detailed review publications outlining the upcoming CyanoSource resource in Microorganisms (https://doi.org/10.3390/microorganisms7100409). Two further review papers are currently in the second round of review and should be accepted shortly.
Exploitation Route CyanoSource will become a key resource for cyanobacterial researchers and algal biotechnology around the globe.
Sectors Education,Environment,Manufacturing, including Industrial Biotechology

URL http://mccormick.bio.ed.ac.uk/