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.

picocyanobacterium prochlorococcus是全球海洋生态系统中最小，最丰富的光合作用组织，占全球碳固定的近10％，大约与全球农田一样多。它的抽象归因于其巨大的遗传多样性。虽然每个细胞包含大约2,000个基因，但核酸群集体具有超过80,000个独特的基因，使这些细胞能够影响广泛的海洋环境。但是，这些基因大多数都是未知的功能。因此，研究人员无法破译其对环境梯度沿不同“生态型”生态学的独特贡献。瓶颈缺乏操纵基因以确定其功能的能力。研究人员将基于他们以前的工作，以优化一组用于核酸核酸基因操纵的方法。开发这些技术是开发甲氯环球菌作为人工光合作用的潜在底盘的关键第一步。波士顿公共图书馆的科学外展活动将设计和实施，并开发了免费的公开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 Labs开始开发一个用于核酸核酸菌群及其近亲海洋Synechococcus的遗传工具包。因为目前尚不清楚核球菌基因组中的哪些位点将是必不可少的，或者将如何有效地构造在基因组中，因此将使用两种宽松的方法来开发prochorococcus的遗传系统。第一个目的将涉及开发一种通过进一步优化用于核酸核酸的CRISPR-CPF1质粒工具，或通过诱导天然，化学，化学物质或电竞争力的线性淘汰盒来进一步优化crispr-cpf1质粒工具，以开发一种基因靶向基因的方法。第二个目的将涉及通过使用自然能力来摄取体外转座子库，或通过使用预组装的定制Transposon Transposon-TN5 Transposase复合物通过电动细胞来产生自然能力，或者通过使用自然能力来生成转座子库。总的来说，这项工作将进一步发展为跨阶段系统生物学的模型系统，并允许研究人员表征这些未知功能的基因如何有助于地球海洋中核酸菌群的全球成功。这些研究的结果将在科学会议上介绍，并在同行评审的科学期刊上发表。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响评估标准，被认为值得通过评估来提供支持。