IOS EDGE: Development of genetic tools for the dominant phototroph in the sea

IOS EDGE：开发海洋中优势光养生物的遗传工具

基本信息

批准号：
1645061
负责人：
Sallie Chisholm
金额：
$ 89万
依托单位：
Massachusetts Institute of Technology
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2022-04-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1645061&HistoricalAwards=false
关键词：
IOS EDGE Development genetic tools

项目摘要

Phytoplankton are microscopic plants that form the base of ocean food chains and collectively do half of the photosynthesis on Earth, equal to all the plants on land. Prochlorococcus is the single most abundant phytoplankton species in the global oceans and is responsible for producing an estimated 4 billion tons of fixed carbon each year, which is approximately the same productivity as global croplands. In addition they are the smallest and most efficient photosynthesizers designed by nature, relying only on sunlight, CO2 and essential nutrients for growth. For the past 25 years, research on this minimal prototroph has led to a growing understanding of its features across all scales of biological organization, from the genome to the global ecosystem. This fascinating microbe has also offered a great opportunity to foster public interest on topics such as ocean ecology, biodiversity and genomics. It attracts the attention of science journalists, and serves as an "ambassador" for microorganisms - exemplifying their important role in planetary maintenance. The one thing that is missing from the Prochlorococcus model system tool kit is the ability to do genetics. This bacterium has resisted all genetic manipulation attempts to date, which has significantly hampered the rate of discovery. The goal of this project is to directly address this limitation by developing an efficient genetic system for Prochlorococcus using both traditional and state-of-the-art tools developed specifically for this project.Research on Prochlorococcus has expanded steadily over the past 25 years, as more and more strains have been brought into culture, and their distribution in the global oceans has been mapped extensively. The PIs now have 100s of Prochlorococcus cultures in the lab, an extensive amount of transcriptomics and proteomics data, and they continue to accumulate genomic data from cultures and wild populations. However each new Prochlorococcus strain, or single cell sequenced, adds roughly 100 entirely new genes to the Prochlorococcus pangenome, the vast majority of which are of unknown function. The development of a genetic system is necessary for identifying the functions of these unknown genes and testing hypotheses involving those with putative functions. This project notably involves a high throughput microfluidic electroporation technology to transform recalcitrant microbes orders of magnitude faster than traditional approaches. A global mutagenesis (Transposon-based tools) and targeted mutagenesis (CRISPR-based tools) strategy will be implemented for Prochlorococcus, while optimizing the microfluidic electroporation technology to its full potential. As proof-of-concept, each tool will be tested in its ability to efficiently reveal gene functions by using our current known repertoire of nutrient acquisition genes in Prochlorococcus.

浮游植物是微观植物，构成了海洋食品链的基础，并统称地球上光合作用的一半，等于土地上的所有植物。 ProChorococcus是全球海洋中最丰富的浮游植物物种，负责每年产生约40亿吨的固定碳，与全球农田的生产率大致相同。此外，它们是由自然设计的最小，最有效的光合作用者，仅依靠阳光，二氧化碳和必需营养素才能生长。在过去的25年中，对这一最小原始植物的研究使从基因组到全球生态系统的生物组织的所有范围都有越来越多的理解。这种迷人的微生物还提供了一个很好的机会，可以在海洋生态学，生物多样性和基因组学等主题上培养公众利益。它吸引了科学记者的注意，并充当微生物的“大使” - 体现了它们在行星维护中的重要作用。 ProChorococcus模型系统工具套件中缺少的一件事是进行遗传学的能力。该细菌已经抵抗了所有遗传操作尝试的尝试，这严重阻碍了发现率。该项目的目的是通过使用专门针对该项目开发的传统和最先进的工具来开发有效的遗传系统来直接解决这一限制。在过去的25年中，对Prochlorococcus进行了稳步扩展，因为在过去的25年中，越来越多的菌株已将其在全球海洋中的分布带入了越来越多的菌株。 PIS现在在实验室中具有100次的核球菌培养物，大量的转录组学和蛋白质组学数据，它们继续从培养物和野生种群中积累基因组数据。但是，每个新的核球菌菌株或测序的单细胞菌株都会向pan氯环球菌添加大约100个全新的基因，其中绝大多数具有未知功能。遗传系统的发展对于识别这些未知基因的功能以及涉及具有推定功能的假设的假设是必要的。该项目特别涉及高吞吐量微流体电穿孔技术，以比传统方法快地改变顽固的微生物订单。将针对氯环菌实施全球诱变（基于转座的工具）和靶向诱变（基于CRISPR的工具）策略，同时优化微流体电动技术的全部潜力。作为概念验证，将通过使用当前已知的养分获得基因的库存库来测试每个工具，以其有效揭示基因功能的能力进行测试。