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

蓝藻是一类进化上古老而丰富的产氧光合细菌，它们加起来占全球固碳总量的25%。在一个目前价值5亿英镑的行业，有几种植物被用于食品、营养食品和染料生产。其他是合成药品、工业化学品和生物燃料的潜在平台。它们特别适合于植物药物的生产，过去十年这一市场的价值超过了GB 20b，因为蓝藻和植物之间的许多生理和生化特征是保守的。尽管蓝藻很重要，但我们对蓝藻生物学的许多关键方面的了解是有限的，这阻碍了我们对蓝藻作为有效生物技术平台的基本了解和发展。用于理解基因功能的关键技术是突变的产生和特征，在突变中，特定的基因或感兴趣的基因被删除。然而，蓝藻突变体的产生是耗时、昂贵的，并且需要特定的专业知识，并不是所有的研究小组都能获得。此外，研究小组使用的不同亚株可能在基因或表型水平上有所不同。这导致了不同研究之间结果的重复性问题，这越来越被认为是科学研究中的一个问题。此外，未能公布结果可能会导致实验室产生(或试图产生)相同的突变，导致不必要的实验重复和资源浪费。在这个项目中，我们将使用分别位于爱丁堡和诺维奇的两个最近建立的英国DNA铸造厂，它们包含自动化机器人技术，能够快速构建全基因组突变文库。使用这些铸造物，我们将针对模式蓝藻聚胞藻中的3,456个基因。PCC6803(集胞藻)。聚球藻是研究最广泛的蓝藻，在生物技术方面具有许多优势。它可以被基因操纵，在与二氧化碳一起冒泡时迅速生长，并能容忍广泛的环境条件。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球藻，聚球藻自然地进口DNA。聚球藻将通过一种称为同源重组的强大过程进行转化，这将导致一个完整的基因敲除突变体文库。这一资源将极大地帮助研究界进行基因功能研究。这个突变文库的建立还将使我们能够确定哪些基因对于聚球藻在典型的实验室生长条件下的生存是必不可少的。条件性突变体(即需要外部刺激来抑制基因的特殊突变体)将针对无法移除的必要基因构建。在这里，我们将使用铜敏感的启动子，当铜存在时，它会关闭基因。所有的质粒和突变体将通过一个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进行更新。总体而言，这些资源将大大促进蓝藻研究和生物技术应用菌株的开发。

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.