SGER: Assessing Genetic Mechanisms of DNA Repair in Ancient Ice Microbes through Analytical Flow Cytometry, High-Speed Cell Sorting, and Single Cell Genomics

分析流式细胞术和细胞分选方面的技术进步正在迅速改变我们对微生物遗传多样性及其与各种环境系统中功能的关系的理解。流式细胞术和高速分选的原位应用是询问亚群的生理状态和活性以及评估特定基因对细胞功能的重要性的不可或缺的组成部分。我们建议将分析流式细胞术、高速细胞分选和单细胞多重置换扩增(MDA)中的新兴技术应用于100Kyr到8Myr的南极冰样，以评估活细胞与死亡细胞的相对比例，并探索古代冰微生物DNA修复的遗传策略。暴露在宇宙辐射中约8Myr的冰中活微生物的恢复表明，古冰中的微生物亚群具有特别有效的DNA修复机制。鉴于宇宙通量对古代冰样中DNA降解的强烈影响以及DNA有效修复的证据，我们假设DNA损伤的程度随着时间的推移而增加，活的8 Myr老细菌具有特别有效和新颖的DNA修复机制，我们可以获得其遗传特征。这项研究的目标是从地球上最古老的冰层中获得用于活细菌修复DNA的基因库。使用以前不适用于古代冰上微生物的分析技术。因此，拟议的研究是一项具有潜在高风险的“探索性”努力。通过我们的分析，很可能会发现新的遗传能力，也许会为生命在地质时期的生存提供关键的分子洞察力，也许还会在其他星球上提供。鉴于标准的流式细胞仪的使用和基因组分析的日益可负担性，我们的研究战略可以转移到科学界，并应该为冰微生物的研究提供关键优势。这个项目将允许年轻的PI继续专业发展，并提供一个机会，让不同教育背景的研究人员(本科生、研究生、技术员、博士后和教职员工)可以互动和发展。

Technological advances in analytical flow cytometry and cell sorting are rapidly revolutionizing our understanding of microbial genetic diversity and its relationship to function in widely varied environmental systems. The in situ application of flow cytometry and high-speed sorting is now an indispensable component for interrogation of the physiological state and activity of subpopulations and assessment of the importance of specific genes to cell function. We propose to apply emerging techniques in analytical flow cytometry, high-speed cell sorting, and single-cell multiple displacement amplification (MDA) to Antarctic ice samples ranging 100 Kyr to 8 Myr in order to assess the relative proportion of viable vs. dead cells and to explore genetic strategies of DNA repair in ancient ice microbes. Recovery of viable microbes within ice exposed to cosmic radiation for ~8 Myr suggests that subpopulations of microbes within ancient ice possess particularly efficient DNA repair mechanisms. Given the strong influence of cosmic flux on DNA degradation in ancient ice samples and evidence of effective DNA repair, we hypothesize that the degree of DNA damage increases with time and that viable 8 Myr old bacteria possess particularly effective and novel DNA repair mechanisms for which we can obtain genetic signatures. The research goal is to obtain a library of genes used for DNA repair by viable bacteria from the oldest ice on earth. Using analytical techniques not previously applied to ancient ice microbes. Thus, the proposed research is an 'exploratory' endeavor with potentially high risk. It is likely that novel genetic capabilities will be discovered through our analyses, perhaps providing key molecular insight into the survival of life over geological time, and perhaps on other planets. Given the use of standard flow cytometry instrumentation and the increasing affordability of genome analyses, our research strategy is transferable to the scientific community and should offer key advantages to the study of ice microbes. This project will allow for continued professional development of a young PI and provide an opportunity whereby researchers with different educational backgrounds (undergraduate students, graduate students, technician, post-docs, and faculty) can interact and develop.