SBIR PHASE I: Cloning and Automated Screening of Genes Encoding Cold-Adapted Lipases From Bacteria Inhabiting Lipid-Rich Whale Skeletons

小行星9561829 这个小企业创新研究第一阶段项目将确定使用新的克隆和自动筛选方法来组装编码冷适应脂肪酶的基因库的可行性。 该方法是基于“环境文库”的构建，这些文库代表了保存在克隆载体中的天然原核生物的集体基因组，这些克隆载体可以在大肠杆菌中繁殖。coli或其它合适的宿主。 由于克隆的DNA最初直接从环境样品中提取，因此文库不限于可以在纯培养物中生长的小部分原核生物，也不偏向于少数快速生长的物种。 然后使用荧光连接的底物和高通量机器人技术筛选这些文库，以鉴定表达感兴趣活性的克隆。 此外，将使用专门的克隆载体，其允许快速鉴定克隆的DNA片段，所述克隆的DNA片段含有靶向用于输出的基因产物的信号序列。 这些信号经常先于编码水解酶如脂肪酶的基因。 这些方法将用于筛选从独特环境中提取的DNA构建的环境文库：深海盆地中富含脂质的鲸鱼骨骼的细菌垫。 初步的研究表明，这些地点拥有一个多样化的细菌群落，这些细菌群落依靠鲸鱼骨骼多年来渗出的大量复合脂质生存。 通过将环境文库方法与高通量自动化筛选相结合，将筛选来自鲸骨细菌的数十万个重组克隆的脂肪酶活性。 编码在低温下显示高活性但在中等温度下保持稳定的脂肪酶的候选基因将被过表达以用于作为洗涤剂添加剂的评估。 此外，还将评估发现的其他独特脂肪酶在其他工业过程中的用途，包括食品和饮料、制药、废物处理系统、化学和医疗行业。

9561829 Stein This Small Business Innovation Research Phase I project will determine the feasibility of using a novel cloning and automated screening approach to assemble a library of genes encoding cold-adapted lipase. The approach is based on the construction of "environmental libraries" which represent the collective genomes of naturally occurring prokaryotes archived in cloning vectors that can be propagated in E. coli or other suitable host. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture nor are they biased towards a few rapidly growing species. These libraries are then screened using fluor-linked substrates and high throughput robotics to identify clones expressing the activity of interest. In addition, specialized cloning vectors will be employed that allow the rapid identification of cloned DNA fragments containing signal sequences which target the gene product for export. Such signals frequently precede genes encoding hydrolytic enzymes such as lipases. These approaches will be used to screen environmental libraries constructed from DNA extracted from a unique environment: the bacterial mats encrusting lipid-rich whale skeletons in deep sea basins. Preliminary work suggests that these sites harbor a diverse bacterial community that subsist on the copious amounts of complex lipids that exude from the whale bones over a period of many years. By coupling the environmental library approach to high throughput automated screening, hundreds of thousands of recombinant clones from the whale bone bacteria will be screened for lipase activity. Candidate genes encoding lipases that display high activity at low temperatures yet remain stable at moderate temperatures will be overexpressed for evaluation as detergent additives. In addition, other unique lipases found will be evaluated for use in other industrial processes which include thos e in food and beverage, pharmaceutical, waste systems, chemical and medical industries.