Desired Number of Participants: 1
Background Information: Despite advances in the specificity and sensitivity of molecular biological technologies, the efficient recovery of DNA from low-biomass samples remains extremely challenging. Optimal methods to extract these biomolecules should 1) achieve the greatest total yield; 2) reflect comprehensive microbial diversity of the sampled environment; and 3) assert reproducible results. For an in-depth assessment of the wide spectrum of microorganisms present in the low-biomass spacecraft assembly clean room environment, technologies facilitating efficient collection, sample processing, and analysis are needed.
Project Description: A homogenous mixture of equal concentrations of several microbial lineages having significant relevance to planetary protection (bacteria, archaea, and fungi; aerobes and anaerobes; cells and spores; rods and cocci) will be prepared. Suitable aliquots of this "model" community will then characterized using a parallel set of downstream molecular analyses which would reveal the level of microbial DNA, extracellular DNA, dissolved organic matter, and particulate non-microbial substances present in the community. Subsamples of this model community will be dried on stainless steel metal surfaces, and procedures targeting the efficient removal and recovery of community member DNAs will be evaluated. The collection and release of genetic materials from various sampling materials/devices will be compared. Several automated nucleic acid extraction methods should be assessed for both total DNA yield and conservation of microbial community structure. Universal small subunit rrn Q-PCR, species-specific Q-PCR, and DNA microarray methodologies will be used in concert to estimate the recovery of both individual members, and the community as a whole. Results of this study will enable consideration of future planetary protection policy amendments based on modern molecular methods.
Web or Literature References: 20. La Duc, M. T., S. Osman, and K. Venkateswaran. 2009. Comparative analysis of methods for the purification of DNA from low-biomass samples based on total yield and conserved microbial diversity. J Rapid Meth Auto Microbiol 17:350-368.
Suggested/required Background/skills, Courses: Microbiology, Molecular Biology, Bioinfomratics, Biochemistry class