Engineering a DNA Nanobarcode to Track Bacterial Population in Agriculturally Important Microbial Environments

NON-TECHNICAL SUMMARY: Due to the high level of diversity in the microbial flora and the intensive temporal changes in temperature, pH and moisture content, aerated composting represents a more complex microbial community than many other microbial ecosystems. Conventional and current methods have limited success in tracking its temporal changes of bacterial population driven by aerobic metabolism. We believe that nanoscale science and engineering, especially the proposed, DNA-based nanobarcode system may provide an exciting and novel opportunity to overcome the difficulties of tracking microbial population dynamics.

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APPROACH: Built upon our expertise and success in nucleic acid engineering, the proposed research will lead to the construction of a variety of novel, nanoscale shapes of DNA (branched DNA including Y-shaped DNA). Tree-shaped, dendrimer-like DNA (DL-DNA) will then be synthesized from these different shapes, and their sizes, patterns, and reactive groups will be precisely controlled. Because DL-DNA is multivalent, monodisperse and anisotropic, different fluorodyes can be conjugated onto each branch of DL-DNA. Thus, this designer DNA will be used as scaffolding for the nanobarcodes. The barcodes will be decoded by the differences in color intensities (i.e., color ratios). These DNA barcodes are in essence 'soft DNA chips' with almost unlimited coding capacity. There are two key components to the proposed project: one, the development of the desired fundamental nanotechnology: the coding/decoding system for nanobarcodes; and two, the assessment of the utility of DNA nanobarcodes in an agriculturally important environment by detecting multiple microbial players in a micro-ecological community of an aerated composting bench scale reactor. Technologies developed in these studies may provide us with novel DNA barcode systems that are rationally designed, assembled at nanoscale, and customized for different agriculture and food system settings.
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PROGRESS: 2005/09 TO 2008/08 <BR>
OUTPUTS: For the 1st year of the project, we have achieved progresses in developing the tracking system with DNA nanobarcodes. In particular we have been able to detect up to 10 molecular targets simultaneously. We have also broadened this technology to blot based as well as detecting in situ single copy genes. A portable detection device is being developed for field usage. For the 2nd year of the project, we have further expanded our DNA nanobarcode system such that these DNA molecules can be engineered into anisotropic and addressable molecules. Signal amplifying is achieved within only one such DNA molecule. We have also achieved in situ detection of a single copy gene within a single bacteria without PCR. Furthermore, we have assembled a prototype device that can be used as a portable detector for multiplexed pathogen sensing. We graduated 1 PhD student and 1 maser student. Overall, our long-term goal of this project is to manipulate DNA in the true engineering sense (nucleic acid engineering) in order to utilize it as a generic instead of a genetic material. In particular, this project focuses on engineering a DNA nanobarcode and then assessing its utility in molecular ecology studies. We have developed DNA nanobarcoding technology along with a portable detector device; combined these outputs are becoming efficient sensing tool that connects nanotechnology with eco-scale microbial analysis by quantitatively tracking dynamic changes in complex microbial communities. We are also applying our nanobarcoding technology beyond molecular ecology studies. For example, the nanobarcode detecting system are being extended to food safety studies as well as clinical diagnoses where multiple pathogens, toxins and other microorganisms can be simultaneously detected. <BR> PARTICIPANTS: Y. Li, S. Um, H. Funabashi, N. Park, W. Cheng, J. Lee, T. Tran, H. Rho, M. Campolongo, E. Yang, P. Tu, M. Dadlani, Y. Cu, S. Kwon, L. Walker, D. Luo <BR> TARGET AUDIENCES: Research groups (scientists and engineers), potential farmers who need fast, inexpensive, and multiplexed detections.
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IMPACT: 2005/09 TO 2008/08<BR>
The nanobarcoding technology developed with this project will impact pathogen detection in general and bacterial tracking in particular. DNA nanobarcode system is a platform, multiplexed detection technology that has found applications in a variety of fields including agriculture, ecology, hydrology, along with medicine. The portable device is powered by batteries and interfaced with laptop computers; it has a potential to be used in the field for the multiplexed detection of different pathogens simultaneously. In summary, the research demonstrated for the first time a multiplexed detection scheme using color ratio intensity and branched DNA molecules. A portable detecting device has been developed and will enable both researchers and end users (e.g. farmers) to detect and track multiple species (e.g. bacteria) in the field. The combined technology can also be used to monitor composter environment, food safety, environment pollutions, etc. <BR> We have graduated two PhD students and 2 MS students. Numerous papers have been published (see refs); notably we have had four Nature-series papers published partially supported by this project.