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

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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.