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

原绿球藻是全球海洋生态系统中最小、数量最多的光合作用生物，贡献了全球近10%的碳固定，大致相当于全球农田的碳固定量。它的丰富归功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，但原绿球藻集体拥有超过80,000个独特的基因，使这些细胞能够在各种海洋环境中生存。然而，大多数这些基因的功能是未知的；因此，研究人员无法破译它们对沿环境梯度的不同“生态型”生态的独特贡献。瓶颈是缺乏操纵基因以确定其功能的能力。研究人员将在他们之前的工作的基础上，优化一套原绿球藻基因操作的方法。开发这些技术是开发原绿球藻作为人工光合作用的潜在基础的关键的第一步。将在波士顿公共图书馆设计和实施以蓝藻遗传学为重点的科学推广活动，以及开发一个免费的、公开可用的生物信息学可视化模块，向学生讲授细菌水平基因转移事件。最近的EDGE资助使奇泽姆和伯顿实验室开始开发原绿球藻及其近亲海洋聚球菌的基因工具包。由于尚不清楚原绿球藻基因组中哪些位点是必需的，以及构建物如何有效地整合到基因组中，因此将采用双管齐下的方法来开发原绿球藻的遗传系统。第一个目标是通过进一步优化原绿球藻CRISPR-Cpf1质粒工具，或通过诱导自然、化学或电致吸收线性敲除盒，开发一种靶向敲除原绿球藻染色体中基因的方法。第二个目标将涉及在原绿球藻中产生转座子文库，通过使用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转座子酶复合物的电穿孔细胞产生体内转座子文库。总的来说，这项工作将进一步发展原绿球藻作为跨尺度系统生物学的模型系统，并允许研究人员描述这些未知功能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，并在同行评议的科学期刊上发表。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.