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Er-2312 Serdp FINAL REPORT Advanced Environmental Molecular Diagnostics to Assess, Monitor, and Predict Microbial Activities at Complicated Chlorinated Solvent Sites SERDP Project ER-2312 DECEMBER 2020 Frank E. Löffler*, Ph.D. (Principal Investigator) Kirsti Ritalahti, Ph.D. (Co-Principal Investigator) University of Tennessee Robert L. Hettich*, Ph.D. (Co-Principal Investigator) Karuna Chourey, Ph.D. (Co-Principal Investigator) Oak Ridge National Laboratory Research Team Members: Jun Yan*, Ph.D. (University of Tennessee) Burcu Şimşir, Ph.D. (University of Tennessee) Yi Yang, Ph.D. (University of Tennessee) Fadime Kara Murdoch*, Ph.D. (University of Tennessee) Gao Chen*, Ph.D. (University of Tennessee) Devrim Kaya, Ph.D. (University of Tennessee) Cynthia M. Swift*, M.S. (University of Tennessee) Ivan Villalobos-Solis*, B.S. (University of Tennessee) * Contributed to the preparation of the Final Report Distribution Statement A This report was prepared under contract to the Department of Defense Strategic Environmental Research and Development Program (SERDP). The publication of this report does not indicate endorsement by the Department of Defense, nor should the contents be construed as reflecting the official policy or position of the Department of Defense. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement or recommendation by the Department of Defense. 2 Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202- 4302. 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PERFORMING ORGANIZATION REPORT 8QLYHUVLW\RI7HQQHVVHH 2DN5LGJH1DWLRQDO/DERUDWRU\ NUMBER .QR[YLOOH, 71 2DN5LGJH71 USA 86$ (5 9.SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10.SPONSOR/MONITOR’S ACRONYM(S) 6(5'3 6WUDWHJLF(QYLURQPHQWDO5HVHDUFKDQG'HYHORSPHQW3URJUDP 4800 Mark Center Drive, Suite ) 11. SPONSOR/MONITOR’S REPORT Alexandria, VA 22350-3605 NUMBER(S) (5 12. DISTRIBUTION / AVAILABILITY STATEMENT 'LVWULEXWLRQ$Approved for public release; distribution is unlimited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a. NAME OF RESPONSIBLE PERSON OF ABSTRACT OF PAGES )UDQN(/|IIOHU a. REPORT b. ABSTRACT c. THIS PAGE 19b. TELEPHONE NUMBER (include area 81&/$66 81&/$66 code) 81&/$66 81&/$66 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Table of Contents #! )$ #'%! $ $%!&#$ $%!$ $%#% A.1 Objectives. ......................................................................................................................... 18 A.2 Technical Approach. .......................................................................................................... 18 A.3 Results. ............................................................................................................................... 19 A.4 Benefits. ............................................................................................................................. 19 #!& High-throughput OpenArray® qPCR ....................................................................................... 25 Environmental proteomics ........................................................................................................ 26 Novel microorganisms involved in the degradation of chlorinated solvents ............................ 28 Organism- and process-specific biomarkers ............................................................................. 29 Response of organohalide-respiring bacteria to perturbations ................................................. 31 Database mining ....................................................................................................................... 32 %!$ Task 1: RD-qChip – Design qPCR assays and validate with defined samples ........................ 34 Selection of gene targets ....................................................................................................... 34 Primer and probe design for qPCR ...................................................................................... 34 Reverse transcription qPCR (RT-qPCR) .............................................................................. 35 qPCR assay validation .......................................................................................................... 36 Standard curve preparation .................................................................................................. 36 OpenArray® plate layout and design ................................................................................... 37 Sample preparation for OpenArray® plate .......................................................................... 40 OpenArray® qPCR ............................................................................................................... 40 Validation of RD-qChip v1 ................................................................................................... 40 Validation of RD-qChip v2 ................................................................................................... 40 Task 2: Proteomics – develop proteomics pipeline and validate with defined samples ........... 40 In silico analysis to evaluate the uniqueness of peptidomes of Dhc strains ......................... 40 RDase phylogenetic tree construction .................................................................................. 41 Dehalococcoides mccartyi (Dhc) cultures and growth conditions ....................................... 41 Task 3: Novel microbes ............................................................................................................ 41 Source material and growth conditions ................................................................................ 41 DNA extraction, PCR and amplicon sequencing .................................................................. 42 Quantitative real-time PCR (qPCR) ..................................................................................... 42 Dhc and Dhgm abundances in groundwater impacted with chlorinated solvents ............... 43 3 Task 4: Identification of novel biomarkers for the degradation of chlorinated solvents .......... 44 Discovery of a novel biomarker for VC reductive dechlorination in mixed culture GP ...... 44 Metagenome sequencing and comparative analyses......................................................... 44 Genome binning and annotation ....................................................................................... 44 Proteomics analysis ........................................................................................................... 45 Discovery of a biomarker for reductive dechlorination of 1,2-D ......................................... 45 1,2-D-dechlorinating cultures ........................................................................................... 45 Microcosms and enrichment cultures ............................................................................... 45 DNA Isolation
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