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

我的研究计划旨在发现和表征产生重要藻类代谢物二甲基磺酸丙酸酯(DMSP)的酶(蛋白质催化剂)。DMSP对1)保护细胞的海洋藻类；2)海洋细菌更喜欢DMSP作为硫源；3)在全球生物地球化学硫循环中提供一半的硫；以及4)气候调节，因为DMSP产生的气溶胶形成云。我利用3种互补策略(比较基因组学、细菌功能互补分析和酶纯化)来发现编码DMSP代谢酶的基因。比较基因组学和细菌功能互补分析确定了我们在细菌宿主中以重组酶的形式表达的DMSP候选基因，然后需要进行纯化。可以对天然藻类DMSP酶进行纯化，以使蛋白质测序工具能够揭示编码这些DMSP酶的基因。这些策略都依赖于1)核酸的分离，2)纯化的、高质量的天然和重组酶，3)衍生化和分离恶劣化学品中的酶产物和底物，以确认和研究DMSP酶的功能。我正在申请资金，购买一台带有气雾剂密封、耐化学物质转子的冷藏微型离心机，它立即需要用来更换一个已有23年历史、无法操作和不可修复的装置。该离心机将用于纯化核酸、DMSP代谢物，帮助研究藻类提取物中的天然DMSP酶或转基因细菌提取物中的重组酶。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基因鉴定将促进对DMSP产生在海藻中的广泛分布的了解，并可使预测藻类DMSP产生对未来海洋条件的反应的研究成为可能。这些DMSP酶也可以被引入作物中，以赋予它们DMSP的细胞保护作用，从而培育出抗逆性强的作物。我所要求的离心机将被我的博士后(2名)、硕士(2名)和本科生(15名以上)以及技术人员(2名)大量使用，为加拿大的下一代科学家提供出色的培训。

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.