Project 004 Description:This project will be broken into two main phases: Phase 1; evaluate pretreatment of samples; and Phase 2; evaluate optimum conditions for enzymatic
cell lysis. Each phase is designed to lead to a specific deliverable that will inform a major step of the nucleic acid extraction protocol. As such, completion
of both phases is not required for success and students will learn to develop experimental matrices, develop sterile technique for working with low
biomass, and learn to extract nucleic acid from Mars analog samples within each phase. In all phases samples will be run in triplicate and with positive
(using a mock microbial community supplied by Zymo) and negative (using nucleic acid free glass beads) controls. It is expected that 10 hours/week will
be required to complete the project. Phase 1: Evaluate pretreatment of soils. Microbial strains isolated from the AHI springs are known to entomb
themselves in carbonate minerals, being hypersaline springs, these samples also have inherently high salt concentrations. These characteristics are
challenging for downstream nucleic acid extraction. Cells entombed in carbonate may not be lysed and therefore their DNA will not be extracted, and
they will be missing from the final diversity evaluation, ie, extraction will be biased against cells entombed in carbonate. Pretreatment with acid digestion
will dissolve carbonate minerals, exposing these cells to downstream extraction. High salt concentrations (and now acidic conditions) interfere with
downstream enzymatic digests and DNA binding to extraction columns. To mitigate this, sample washes with phosphate buffered saline (PBS) will dilute
salts and neutralize acid. Students will evaluate the effect of different concentrations of dilute HCl and different osmotic strengths of PBS in sequential
sample pretreatment steps prior to following the established protocol for Qiagen PowerLyzer Power Soil DNA extraction kits on the recovered microbial
diversity. Phase 2: evaluate optimum conditions for enzymatic cell lysis. To capture recalcitrant cells, multiple methods of cell digestion and lysis are
required. In phase 1, students evaluate the efficacy of acid (chemical) digestion. In phase 2, students will evaluate enzymatic digestion. There are two
formulations of enzymatic solutions optimized for low biomass, litho(auto)trophic communities: Metapolyzyme, which contains a collection of enzymes
that digest cell walls and Exopolyzyme, which contains a collection of enzymes that digest extrapolymeric substances, or the recalcitrant material that
adheres cells to their physical rock substrates. Each specific enzyme in both mixes has a maximum efficiency at a specific optimum pH and temperature
combination. Published literature protocols use an average temperature and pH, and different sample types may benefit from optimizing these
parameters. Again, using AHI spring samples and the established protocol for Qiagen PowerLyzer Power Soil DNA extraction kits, students will develop
experimental matrices to evaluate the effect different temperature and pH combinations, optimized for individual enzymes within each mix, have on the
recovered microbial diversity