Collaborative Research: New Medium for DNA Separation of Microbial Communities

该奖项是根据2009年《美国复苏与再投资法案》(Public Law 111-5)资助的。这项由化学与生物分离计划授予的NSF奖项支持伦斯勒理工学院(RPI)的Linda B.McGown教授和佛罗里达国际大学(FIU)的DeEtta K.Mills教授分别致力于提高DNA图谱方法揭示微生物群落真实多样性的能力。人们越来越认识到复杂的微生物群落和生物膜在环境、工业、医疗、农业和市政应用的工程系统中的重要性，这就产生了对表征这些复杂生命系统的新方法和技术的需求。量化群落内微生物的数量和类型及其对环境压力的反应的能力，对于理解任何包含微生物群落的自然或工程生态系统的结构和功能是至关重要的。由于微生物种群在远离典型人类感知的时空尺度上运行，过去很难研究它们在复杂系统行为中的作用。这已成为分析化学和生物分离领域的前沿挑战。研究微生物群落及其对环境刺激的动态相互作用和反应的最有效方法之一是通过检查它们的DNA图谱或元基因组。然而，现有方法的群落DNA图谱的有效性是有限的，因为分类上不相关的生物可能产生相同长度的DNA，导致低估了群落的真正多样性。然后必须对相同长度的DNA进行测序，以揭示图谱中隐藏的多样性，并获得样本群落的真实多样性。本项目研究一种新的凝胶介质，它是通过鸟苷化合物的自结合形成的，以拆分相同长度的DNA和不同的序列。将新型凝胶与毛细管凝胶电泳中的传统筛选凝胶相结合，将在群落DNA的长度和序列上实现前所未有的分辨率，这将揭示以前被掩盖的群落多样性，而不必对微生物群落的整个元基因组进行测序，从而与目前的方法相比大大减少了时间和成本。RPI的研究将由一名研究生、一名本科生和两名高中生进行，他们将通过RPI的科学技术进入计划(STEP)每年被招募。STEP让地区初中和高中中代表性不足和经济困难的学生参加活动，旨在培养解决问题的技能和进入大专学校之前的广泛知识基础。在少数民族院校FIU，这项研究将由两名本科生进行。在该项目的暑期学期期间，两所院校的学生将进行实习/实验室交流。FIU的学生将在RPI学习4-6周的分离技术，McGown的学生将前往FIU，在那里他们将学习分子技术，如聚合酶链式反应、定量聚合酶链式反应和DNA提取。这次交流将极大地提升这些学生群体的知识深度和深度。所有数据的合并、合成和传播将是国际和平倡议、共同国际和平倡议及其学生共同努力的结果。这些跨学科的努力将使信息在科学领域得到更大的传播，并跨越到工程的关键领域。分子生物学和分离科学的融合是麦高恩和米尔斯实验室当前工作的自然发展。通过为少数民族研究生和本科生的知识发展和辅导提供支持，这种研究经验将通过两所机构之间的实验室互动和学生交流来加强和拓宽他们的知识基础。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).This NSF award by the Chemical and Biological Separations program supports work by Professors Linda B. McGown and DeEtta K. Mills at Rensselaer Polytechnic Institute (RPI) and Florida International University (FIU), respectively, to improve the ability of DNA profiling methods to reveal the true diversity of microbial communities. The growing recognition of the importance of complex microbial communities and biofilms in engineered systems for environmental, industrial, medicinal, agricultural and municipal applications has created a demand for new approaches and techniques for the characterization of these complex living systems. The ability to quantify the number and types of microorganisms within a community and its response to environmental stressors is fundamental to the understanding of the structure and function of any natural or engineered ecosystem containing microbial communities. Because microbial populations operate at spatio-temporal scales far removed from typical human perception, it has been difficult in the past to investigate their role in complex system behavior. This has emerged as a challenge at the forefront of analytical chemistry and bioseparation. One of the most powerful approaches for profiling microbial communities and understanding their dynamic interactions and responses to environmental stimuli is through examination of their DNA profiles, or ?metagenomes?. However, the effectiveness of community DNA profiling with existing methods is limited because taxonomically unrelated organisms may produce the same length DNA, resulting in an underestimation of the true diversity of the community. Same length DNA must then be sequenced in order to reveal the hidden diversity within the profiles and obtain the true diversity of the sampled community.This project investigates a new gel medium that is formed by self-association of guanosine compounds to resolve same length DNA with different sequences. Combining the new gel with conventional sieving gels in capillary gel electrophoresis will lead to unprecedented resolution in length and sequence of community DNA that will reveal previously masked community diversity without having to sequence the entire metagenome of the microbial community, thereby greatly reducing the time and expense compared to current methods. The research at RPI will be conducted by a graduate student, an undergraduate student and two high school students who will be recruited each year through the Science Technology Entry Program (STEP) at RPI. STEP engages underrepresented and economically disadvantaged students in area middle and high schools in activities designed to develop problem-solving skills and a broad knowledge base prior to entry into post-secondary schools. The research at FIU, a minority institution, will be conducted by two undergraduate students. There will be an internship/laboratory exchange of the students from both institutions during the summer semesters of the project. FIU students will spend 4-6 weeks at RPI learning separation techniques and the McGown students will travel to FIU where they will learn molecular techniques such as PCR, qPCR and DNA extractions. This exchange will greatly enhance the breath and depth of knowledge for these student groups. The coalescence, synthesis and dissemination of all data will be a combined effort of the PI, co-PI and their students. These interdisciplinary efforts will result in greater dissemination of information across scientific fields and crossover into critical fields of engineering. The merging of molecular biology and separation sciences evolves as a natural progression of the current work of the McGown and Mills laboratories. By providing support for knowledge development and mentoring of minority graduate and undergraduates, this research experience will strengthen and broaden their knowledge base by the laboratory interactions and student exchange between the two institutions.