Elizabeth Padilla-Crespo is a Ph.D. candidate in theSchool of Biology at the Georgia Institute of Technology. She has a double degree in Industrial Biotechnology and Microbiology from the University of Puerto Rico-Mayaguez and as an undergraduate student, performed research at Harvard Medical School, the DOE Lawrence Berkeley National Laboratory, the University of Wisconsin-Madison and the Georgia Institute of Technology. Ms. Padilla is a NSF-Fellow advised by Dr. Frank Löffler and is currently working on the identification of reductive dehalogenation biomarkers using microarray technology.
Presentation Description Application of an oligonucleotide microarray for reductive dechlorination biomarker identification and process monitoring
Bioremediation has emerged as a productive cleanup strategy at sites impacted with chlorinated solvents (e.g., chlorinated ethenes). Crucial for achieving detoxification are Dehalococcoides bacteria, which possess multiple reductive dehalogenase (RDase) genes. RDase genes are promising targets for monitoring dehalogenation reactions of interest. In order to assign function to putative RDase genes and enhance bioremediation monitoring tools, we designed an oligonucleotide microarray with probes targeting all RDase genes (341) identified in public databases. Also included were probes targeting Dehalococcoides hydrogenase genes (109), 16S rRNA genes and housekeeping genes for normalization. Three oligonucleotide probes (40-50 bp long) were designed per target gene using Oligoarray 2.1 software, and tethered to an epoxy-coated glass slide via C6 amino linkers. An integrated approach combining cultivation of dechlorinating consortia and enumeration of known Dehalococcoides biomarker genes with quantitative real-time PCR (qPCR) tested the functionality of the RDase microarray. Hybridizations used cDNA generated from total RNA extracted from dechlorinating consortia maintained under defined batch and continuous growth (i.e., chemostat) conditions to gain insight into expression of individual RDase genes and to assign functions to Dehalococcoides RDase genes of interest. These experiments revealed that RDase transcription is a dynamic process and that multiple RDase genes are responsive to a single chlorinated substrate suggesting functional redundancy. Ongoing experiments explore the transcriptional changes in response to culture conditions, co-culture interactions and feeding regimes. Experiments also used DNA extracted from contaminated groundwater to monitor RDase gene presence and to gain insight into RDase gene diversity in a subsurface environment undergoing bioremediation. Our findings demonstrate that the RDase array is an excellent tool for identifying process-specific target genes and to monitor reductive dechlorination processes.
