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

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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.