Refrigerated microcentrifuge system for molecular biology, protein biochemistry, and metabolite analysis

用于分子生物学、蛋白质生物化学和代谢物分析的冷冻微量离心机系统

• 批准号: 472746-2015
• 负责人: Waller, Jeffrey
• 金额: $ 0.6万
• 依托单位: Mount Allison University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=564834
• 关键词: Refrigerated; microcentrifuge; system; molecular; biology

My research program seeks to discover and characterize enzymes (protein catalysts) that produce the vital algal metabolite dimethylsulfoniopropionate (DMSP). DMSP is important to 1) marine algae for cellular protection; 2) marine bacteria preferring DMSP as a sulfur source; 3) supplying half the sulfur in the global biogeochemical sulfur cycle; and 4) climate regulation since DMSP-derived aerosols form clouds. I exploit 3 complementary strategies (comparative genomics, bacterial functional complementation assays, and enzyme purification) to discover genes encoding DMSP metabolism enzymes. Comparative genomics and bacterial functional complementation assays identify DMSP gene candidates that we express in bacterial hosts as recombinant enzymes which then need purification. The native algal DMSP enzymes can be purified to enable protein sequencing tools which reveal the genes encoding these DMSP enzymes. These strategies all *depend* on 1) separation of nucleic acids, 2) purified, high-quality native and recombinant enzymes, 3) derivatization and separation of enzyme products and substrates in harsh chemicals to confirm and study DMSP enzyme function. I am applying for funds for a refrigerated microcentrifuge with an aerosol-tight, chemical-resistant rotor that is immediately needed to replace a 23 yr old inoperable and unrepairable unit. This centrifuge will be used to purify nucleic acids, DMSP metabolites, help study the native DMSP enzymes from algal extracts or recombinant enzymes from transgenic bacterial extracts. Purified DMSP enzymes will be characterized with respect to their biochemical structure, regulation, kinetic behaviour, and identity. DMSP gene identification will advance understanding of how widespread DMSP production is in marine algae and can enable studies predicting the response of algal DMSP production to future oceanic conditions. These DMSP enzymes may also be introduced into crop plants to confer DMSP's cellular protection on them, leading to stress-resistant crops. The requested centrifuge will see substantial use by my postdoctoral (2), MSc (2) and undergraduate (15+) students, and technicians (2), giving excellent training for the next generation of scientists in Canada.

我的研究计划旨在发现和表征酶（蛋白质催化剂），产生重要的藻类代谢物二甲基磺基丙酸酯（DMSP）。 DMSP对1）海藻细胞保护; 2）海洋细菌偏好DMSP作为硫源; 3）在全球地球化学硫循环中提供一半的硫; 4）由于DMSP衍生的气溶胶形成云，因此气候调节很重要。 我利用3个互补的策略（比较基因组学，细菌功能互补试验，酶纯化），发现编码DMSP代谢酶的基因。 比较基因组学和细菌功能互补测定鉴定DMSP基因候选物，我们在细菌宿主中表达为重组酶，然后需要纯化。 可以纯化天然藻类DMSP酶以使蛋白质测序工具能够揭示编码这些DMSP酶的基因。 这些策略都 * 取决于 * 1）核酸的分离，2）纯化的高质量天然和重组酶，3）衍生化和分离酶产物和苛刻化学品中的底物，以确认和研究DMSP酶功能。 我正在申请一个冷冻微型离心机的资金，该离心机具有气溶胶密封，耐化学腐蚀的转子，立即需要更换一个23年的旧的无法操作和无法修复的装置。 该离心机将用于纯化核酸、DMSP代谢产物，帮助研究来自藻类提取物的天然DMSP酶或来自转基因细菌提取物的重组酶。 纯化的DMSP酶将根据其生化结构、调节、动力学行为和特性进行表征。 DMSP基因的鉴定将促进了解DMSP生产在海藻中的广泛程度，并可使研究预测海藻DMSP生产对未来海洋条件的反应。这些DMSP酶也可以被引入到作物植物中，以赋予DMSP的细胞保护作用，从而产生抗胁迫作物。我的博士后（2），硕士（2）和本科（15+）学生以及技术人员（2）将大量使用所要求的离心机，为加拿大的下一代科学家提供出色的培训。