{"id":401,"date":"2023-01-19T18:25:46","date_gmt":"2023-01-19T18:25:46","guid":{"rendered":"https:\/\/in.nau.edu\/astro-partnership\/?page_id=401"},"modified":"2025-09-17T21:50:04","modified_gmt":"2025-09-17T21:50:04","slug":"project-0004","status":"publish","type":"page","link":"https:\/\/in.nau.edu\/astro-partnership\/project-0004\/","title":{"rendered":"Project 0004"},"content":{"rendered":"<p><strong>Project 004 Description:<\/strong>This project will be broken into two main phases: Phase 1; evaluate pretreatment of samples; and Phase 2; evaluate optimum conditions for enzymatic<br \/>\ncell 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<br \/>\nof both phases is not required for success and students will learn to develop experimental matrices, develop sterile technique for working with low<br \/>\nbiomass, 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<br \/>\n(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<br \/>\nbe required to complete the project. Phase 1: Evaluate pretreatment of soils. Microbial strains isolated from the AHI springs are known to entomb<br \/>\nthemselves in carbonate minerals, being hypersaline springs, these samples also have inherently high salt concentrations. These characteristics are<br \/>\nchallenging for downstream nucleic acid extraction. Cells entombed in carbonate may not be lysed and therefore their DNA will not be extracted, and<br \/>\nthey will be missing from the final diversity evaluation, ie, extraction will be biased against cells entombed in carbonate. Pretreatment with acid digestion<br \/>\nwill dissolve carbonate minerals, exposing these cells to downstream extraction. High salt concentrations (and now acidic conditions) interfere with<br \/>\ndownstream enzymatic digests and DNA binding to extraction columns. To mitigate this, sample washes with phosphate buffered saline (PBS) will dilute<br \/>\nsalts and neutralize acid. Students will evaluate the effect of different concentrations of dilute HCl and different osmotic strengths of PBS in sequential<br \/>\nsample pretreatment steps prior to following the established protocol for Qiagen PowerLyzer Power Soil DNA extraction kits on the recovered microbial<br \/>\ndiversity. Phase 2: evaluate optimum conditions for enzymatic cell lysis. To capture recalcitrant cells, multiple methods of cell digestion and lysis are<br \/>\nrequired. In phase 1, students evaluate the efficacy of acid (chemical) digestion. In phase 2, students will evaluate enzymatic digestion. There are two<br \/>\nformulations of enzymatic solutions optimized for low biomass, litho(auto)trophic communities: Metapolyzyme, which contains a collection of enzymes<br \/>\nthat digest cell walls and Exopolyzyme, which contains a collection of enzymes that digest extrapolymeric substances, or the recalcitrant material that<br \/>\nadheres cells to their physical rock substrates. Each specific enzyme in both mixes has a maximum efficiency at a specific optimum pH and temperature<br \/>\ncombination. Published literature protocols use an average temperature and pH, and different sample types may benefit from optimizing these<br \/>\nparameters. Again, using AHI spring samples and the established protocol for Qiagen PowerLyzer Power Soil DNA extraction kits, students will develop<br \/>\nexperimental matrices to evaluate the effect different temperature and pH combinations, optimized for individual enzymes within each mix, have on the<br \/>\nrecovered microbial diversity<\/p>\n","protected":false},"excerpt":{"rendered":"<p>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 [&hellip;]<\/p>\n","protected":false},"author":575,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_relevanssi_hide_post":"","_relevanssi_hide_content":"","_relevanssi_pin_for_all":"","_relevanssi_pin_keywords":"","_relevanssi_unpin_keywords":"","_relevanssi_related_keywords":"","_relevanssi_related_include_ids":"","_relevanssi_related_exclude_ids":"","_relevanssi_related_no_append":"","_relevanssi_related_not_related":"","_relevanssi_related_posts":"","_relevanssi_noindex_reason":"","ring_central_script_selection":"","footnotes":""},"class_list":["post-401","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/pages\/401","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/users\/575"}],"replies":[{"embeddable":true,"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/comments?post=401"}],"version-history":[{"count":3,"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/pages\/401\/revisions"}],"predecessor-version":[{"id":505,"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/pages\/401\/revisions\/505"}],"wp:attachment":[{"href":"https:\/\/in.nau.edu\/astro-partnership\/wp-json\/wp\/v2\/media?parent=401"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}