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、数量最多的光合生物，贡献了全球近10%的碳固定-大约相当于全球农田。它的丰富性归因于其巨大的遗传多样性;虽然每个细胞含有约2，000个基因，但原绿球藻集体拥有超过80，000个独特的基因，使这些细胞能够栖息在广泛的海洋环境中。然而，这些基因中的大多数功能未知;因此，研究人员无法破译它们对沿着环境梯度的不同“生态型”生态学的独特贡献。瓶颈是缺乏操纵基因以确定其功能的能力。研究人员将在他们以前的工作基础上优化一套原绿球藻遗传操作的方法。开发这些技术是开发原绿球藻作为人工光合作用潜在底盘的关键第一步。将设计和实施波士顿公共图书馆的科学推广活动，重点是蓝藻遗传学，以及开发一个免费的、公开的生物信息学可视化模块，向学生传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藻的遗传工具包。由于目前还不清楚原绿球藻基因组中的哪些位点是必不可少的，或者构建体将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中的遗传系统。第一个目标将涉及开发一种用于靶向敲除原绿球藻染色体中的基因的方法，通过进一步优化用于原绿球藻的CRISPR-Cpf 1质粒工具或通过诱导线性敲除盒的天然、化学或电感受态摄取。第二个目的将涉及在原绿球藻中产生转座子文库，通过使用用于摄取体外转座子文库的天然感受态，或通过用预组装的定制转座子-Tn 5转座酶复合物电穿孔细胞来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，并使研究人员能够描述这些功能未知的基因如何有助于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发表，并发表在同行评审的科学期刊上。该奖项反映了NSF的法定使命，并被认为值得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来支持。

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.