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

SBIR 第一阶段：从栖息于富含脂质的鲸鱼骨骼中的细菌中克隆和自动筛选编码冷适应脂肪酶的基因

• 批准号: 9561829
• 负责人: Jeffrey Stein
• 金额: $ 7.5万
• 依托单位: Diversa Corporation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9561829&HistoricalAwards=false
• 关键词: SBIR; PHASE; Cloning; Automated; Screening

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.

9561829 Stein 这个小型企业创新研究第一阶段项目将确定使用新型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。 该方法基于“环境文库”的构建，该“环境文库”代表了存档在可在大肠杆菌或其他合适宿主中繁殖的克隆载体中的天然存在的原核生物的集体基因组。 由于克隆的 DNA 最初是直接从环境样本中提取的，因此文库不仅限于可在纯培养物中生长的一小部分原核生物，也不偏向于少数快速生长的物种。 然后使用氟连接底物和高通量机器人筛选这些文库，以识别表达感兴趣活性的克隆。 此外，还将采用专门的克隆载体，以允许快速鉴定含有针对输出基因产物的信号序列的克隆DNA片段。 此类信号经常出现在编码水解酶（例如脂肪酶）的基因之前。 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：深海盆地中包裹着富含脂质的鲸鱼骨骼的细菌垫。 初步研究表明，这些地点蕴藏着多样化的细菌群落，这些细菌群落以鲸鱼骨头中多年以来渗出的大量复杂脂质为生。 通过将环境文库方法与高通量自动筛选相结合，将筛选来自鲸骨细菌的数十万个重组克隆的脂肪酶活性。 编码在低温下表现出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度表达以评估作为洗涤剂添加剂。 此外，还将对发现的其他独特脂肪酶在其他工业过程中的使用进行评估，包括食品和饮料、制药、废物系统、化学和医疗行业。