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

蓝藻是一种进化上古老而丰富的含氧光合细菌，它们共同占全球固碳量的25%。一些品种被用于食品、营养品和染料生产，这个行业目前价值5亿英镑。其他的则是合成药物、工业化学品和生物燃料的潜在平台。它们特别适合生产植物药物，在过去十年中，这个市场价值超过200亿英镑，因为蓝藻和植物之间的许多生理和生化特征是保守的。尽管他们的重要性，我们的蓝藻生物学的许多关键方面的知识是有限的，这阻碍了蓝藻作为有效的生物技术平台的基本理解和发展。用于了解基因功能的一项关键技术是产生和描述突变体，其中特定基因或感兴趣的基因已被删除。然而，蓝藻突变体的产生是耗时的，昂贵的，需要特定的专业知识，并不是所有的研究小组可用。此外，研究小组使用不同的子菌株，可能在基因型或表型水平上有所不同。这导致了不同研究之间结果的可重复性问题，这越来越被认为是科学研究中的一个问题。此外，未能公布结果可能导致实验室产生（或试图产生）相同的突变体，从而导致不必要的实验复制和资源浪费。在这个项目中，我们将使用最近分别位于爱丁堡和诺维奇的两个英国DNA代工厂，它们包含能够快速构建全基因组突变文库的自动化机器人技术。利用晶圆厂，我们将针对模型蓝藻，Synechocystis sp. PCC 6803 （Synechocystis）中的3,456个基因。聚囊藻是研究最广泛的蓝藻，在生物技术方面具有许多优势。它可以通过基因操作，在二氧化碳中迅速生长，并能忍受各种环境条件。为了产生突变体，我们将自动组装基因敲除质粒，这些质粒将被引入聚胞菌，它会自然地输入DNA。聚胞虫将通过一个称为同源重组的强大过程进行转化，这将产生一个完整的敲除突变体库。这一资源将极大地帮助研究界在基因功能的研究。这个突变文库的生成也将使我们能够确定在典型的实验室生长条件下，哪些基因对聚囊虫的生存是必不可少的。条件突变体（即需要外部刺激来抑制基因的特化突变体）将被构建为不能被移除的基本基因。在这里，我们将使用铜敏感启动子，当铜存在时关闭基因。所有的质粒和突变体将通过公共数据库提供给英国和国际研究人员，该数据库将在整个项目中不断更新。总的来说，这些资源将显著推进蓝藻研究和菌株的发展，生物技术的应用。

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.