Developing a Novel Metatranscriptomic Approach for Identifying Biomarkers Directly from Mixed Microbial Communities

1438660De long开发一种新的从混合微生物群落中直接识别生物标志物的超转录组学方法针对功能性生物标志物基因的定量分子生物学分析是一种强大的工具，可用于广泛的应用，包括指导新型环境生物技术的开发和评估自然生态系统对扰动的反应。然而，对于许多系统，目前缺乏相关功能生物标志物基因的知识。使这一问题复杂化的是，目前可用的鉴定可作为生物标志物的基因的方法通常不适合应用于与环境相关的混合微生物群落，因为它们需要分离纯培养物。因此，迫切需要开发可直接应用于混合微生物群落的新型生物标记基因鉴定工具。除了支持环境生物技术的发展外，还将开展教育推广活动，开发基础科学教育的新课程，以提高科学和工程素养的整体水平。将以生物修复和可持续性为主题，开发一个跨学科的基于问题的学习模块。工程研究生研究助理将在初级科学教育专家的指导下，与职前教师一起开发课程。该模块将在科罗拉多州的三所小学进行测试。以问题为基础的学习模块将被开发，以加强21世纪的技能，包括批判性思维，提出问题，解决问题，和创新/创造力的发展。一个工程博士生研究员将通过拟议的项目进行培训。此外，职前教师将接受课程开发方面的实践培训。新课程将通过分析学生的知识、理解能力以及应用和综合单元信息的能力来评估。本研究项目的长期目标是开发基于功能基因的微生物生态学工具，并应用这些工具指导可持续环境生物技术的发展。为了实现这一长期目标，PI和同事们最近采用并验证了一种基于差异基因表达的技术（原核cDNA减法），用于鉴定和测序细菌中的候选生物标记基因；然而，将这种方法应用于混合培养还需要进一步的研究。因此，本应用的目的是利用厌氧邻二甲苯降解产甲烷培养物作为原型模型系统，应用这种原始方法从微生物群落中识别特定的生物降解基因。本研究的目标将通过以下方式实现：1)扩展基于差异基因表达的方法cDNA减法，用于从混合微生物群落中鉴定推定的生物标志物；2)验证通过目标1为模型系统鉴定的功能性生物标志物基因。为了实现目标1，该方法将适应和应用PIs实验室中已经可用的基于差异基因表达的方法，以识别在混合共生培养中厌氧邻二甲苯降解过程中特异性表达的基因。PI还将开发和应用使用比较元转录组学（RNA-seq）的硅cDNA减法方法。为了实现目标2，该方法将通过定量反转录PCR在微观研究中鉴定表达与生物降解性能相关的基因。拟议的工作预计将导致与两种新的生物标志物开发方法相关的整个工作流程的发展，这两种新方法称为减法群落亚转录组学（SCM）和硅SCM，这将使研究人员能够直接从混合微生物群落中开发生物标志物，从而克服迄今为止阻碍从混合和共生微生物群落中有效鉴定生物降解基因的长期障碍。这一贡献是重要的，因为它有望对如何在微生物生态学和环境工程领域开发和应用基于功能基因的工具产生直接和广泛的基础影响。

1438660De LongDeveloping a Novel Metatranscriptomic Approach for Identifying Biomarkers Directly from Mixed Microbial CommunitiesQuantitative molecular biology assays targeting functional biomarker genes are powerful tools that can be used for broad applications including to guide development of novel environmental biotechnologies and to assess the response of natural ecosystems to perturbations. However, for many systems, knowledge of relevant functional biomarker genes is currently lacking. Compounding this problem, currently available methods to identify genes that can serve as biomarkers are not usually suitable for application to environmentally-relevant mixed microbial communities because they require isolation of pure cultures. Thus, a critical need exists to develop novel biomarker gene identification tools that can be applied directly to mixed microbial communities. In addition to supporting the development of environmental biotechnologies, educational outreach will be carried out to develop new curriculum for elementary science education to improve overall levels of science and engineering literacy. An interdisciplinary problem-based learning module will be developed using bioremediation and sustainability as a theme. Engineering graduate research assistants will work with pre-service teachers to develop the curriculum under the guidance of the PI with the support of an elementary science education expert. The module will be tested at three Colorado elementary schools. The problem-based learning module will be developed to enhance development of 21st century skills including critical thinking, question-posing, problem solving, and innovation/creativity. An engineering doctoral student researcher will be trained via the proposed project. Additionally, the pre-service teachers will receive hands-on training in curriculum development. The new curriculum will be evaluated via analysis of pre- and post-test data on student knowledge, comprehension, and abilities to apply and synthesize information from the unit.The long-term goal in this research program is to develop functional gene-based microbial ecology tools and apply these tools to guide development of sustainable environmental biotechnologies. In pursuit of this long-term goal, the PI, with colleagues, has recently adapted and validated a differential gene expression-based technique (prokaryotic cDNA subtraction) for identifying and sequencing candidate biomarker genes in bacteria; however, further research is needed to extend this approach for application to mixed cultures. Therefore, the objective in this application is to apply this original approach to identify specific biodegradation genes from microbial communities using anaerobic o-xylene-degrading methanogenic cultures as a prototype model system. The goal of this research will be achieved by 1) extending differential gene expression-based methodology cDNA Subtraction for identification of putative biomarkers from mixed microbial communities, and 2) validating functional biomarker genes identified via Objective #1 for the model system. To achieve Objective 1, the approach will be to adapt and apply the differential gene expression-based methodology already available in the PIs laboratory to identify genes that are specifically expressed during anaerobic o-xylene degradation in a mixed syntrophic culture. The PI will also develop and apply in silico cDNA Subtraction methodology using comparative metatranscriptomics (RNA-seq). To achieve Objective 2, the approach will be to identify genes for which expression correlates with biodegradation performance in microcosm studies via quantitative reverse-transcription PCR. The proposed work is expected to lead to the development of the entire work flow associated with two novel approaches to biomarker development termed Subtractive Community Metatranscriptomics (SCM) and in silico SCM that will allow researchers to develop biomarkers directly from mixed microbial communities, thus overcoming a longstanding barrier that has to date, prevented efficient identification of biodegradation genes from mixed and syntrophic microbial communities. This contribution is significant because it is expected to have immediate and widespread fundamental implications regarding how functional gene-based tools are developed and applied in the fields of microbial ecology and environmental engineering.