Collaborative Research: EDGE-FGT: Furthering Progress on a Genetic System for the Oceans' Most Abundant Phototrophs

合作研究：EDGE-FGT：海洋最丰富的光养生物遗传系统的进一步进展

• 批准号: 2319334
• 负责人: Briana Burton
• 金额: $ 13.55万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2319334&HistoricalAwards=false
• 关键词: Collaborative; Research; EDGE; FGT; Furthering

The picocyanobacterium Prochlorococcus is the smallest, most numerically abundant photosynthesizing organism in the global ocean ecosystem, contributing to almost 10% of global carbon fixation – roughly as much as global croplands. Its abundance is attributed to its enormous genetic diversity; while each cell contains around 2,000 genes, the Prochlorococcus collective has over 80,000 unique genes, allowing these cells to inhabit a wide range of marine environments. Most of these genes are of unknown function, however; thus, researchers are unable to decipher their unique contribution to the ecology of different “ecotypes” along environmental gradients. The bottleneck has been the lack of ability to manipulate the genes in order to determine their function. The investigators will build upon their previous work to optimize a set of methods for the genetic manipulation of Prochlorococcus. Developing these technologies is a critical first step in developing Prochlorococcus as a potential chassis for artificial photosynthesis. Science outreach events at the Boston Public Library focused on cyanobacteria genetics will be designed and implemented, as well as the development of a free, publicly available bioinformatics visualization module to teach students about bacterial horizontal gene transfer events. Recent EDGE funding has allowed the Chisholm and Burton Labs to begin developing a genetic toolkit for Prochlorococcus and its close relative, marine Synechococcus. Because it is unclear which sites in the Prochlorococcus genome will be essential or how efficiently constructs will be integrated into the genome, a two-pronged approach will be utilized in the development of a genetic system in Prochlorococcus. The first aim will involve development of a method for the targeted knockout of genes in the Prochlorococcus chromosome, by either further optimization of the CRISPR-Cpf1 plasmid tool for Prochlorococcus or by induction of the natural-, chemical-, or electro-competent uptake of a linear knockout cassette. The second aim will involve generation of a transposon library in Prochlorococcus, through the use of either natural competence for the uptake an in vitro transposon library, or through generation of an in vivo transposon library by electroporating cells with a pre-assembled custom transposon-Tn5 transposase complex. Collectively, this work will further develop Prochlorococcus as a model system for cross-scales systems biology and allow researchers to characterize how these genes of unknown function contribute to the global success of Prochlorococcus in Earth’s oceans. Results from these studies will be presented at scientific meetings and published in peer-reviewed scientific journals.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

微微蓝藻原绿球菌是全球海洋生态系统中最小、数量最多的光合生物，贡献了近 10% 的全球碳固定——其丰富程度归因于其巨大的遗传多样性，而每个细胞都含有大约 10% 的碳固定量。原绿球藻共有 2,000 个基因，拥有超过 80,000 个独特基因，使这些细胞能够栖息在广泛的海洋环境中，其中大多数基因都是未知的。然而，研究人员无法解读它们对不同“生态型”生态的独特贡献，而研究人员将缺乏操纵基因以确定其功能的能力。他们之前的工作是优化一套原绿球菌的基因操作方法，开发这些技术是原绿球菌作为正在发展的波士顿公共图书馆的人工光合作用的潜在基础的关键的第一步。将设计和实施蓝藻遗传学，以及开发免费的、公开的生物信息学可视化模块，以向学生传授细菌水平基因转移事件。最近的 EDGE 资金使 Chisholm 和 Burton 实验室能够开始开发原绿球藻的遗传工具包。及其近亲海洋聚球藻，因为尚不清楚原绿球藻基因组中的哪些位点是必需的，或者构建体将如何有效地整合到基因组中，因此需要采取两方面的措施。该方法将用于开发原绿球藻遗传系统，第一个目标将涉及通过进一步优化原绿球藻的 CRISPR-Cpf1 质粒工具或通过诱导来开发一种靶向敲除原绿球藻染色体中的基因的方法。线性敲除盒的自然、化学或电感受态摄取的第二个目标将涉及通过使用原绿球藻中的转座子文库。摄取体外转座子文库的天然能力，或通过使用预组装的定制转座子-Tn5转座酶复合物电穿孔细胞来生成体内转座子文库。总的来说，这项工作将进一步开发原绿球藻作为模型系统。跨尺度系统生物学，使研究人员能够表征这些功能未知的基因如何促进原绿球藻在地球海洋中的全球成功，这些研究的结果将在科学会议上提出并发表在《科学》杂志上。同行评审的科学期刊。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响评审标准进行评估，被认为值得支持。