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Nano-screening of marine symbiotic consortium for useful bio-materials and their analyses

海洋共生体有用生物材料的纳米筛选及其分析

基本信息

批准号：
15360437
负责人：
TAKEYAMA Haruko
金额：
$ 9.54万
依托单位：
National University Corporation Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15360437/
关键词：
Marine symbiotic consortium Nano-sized beads Symbiotic Associated bacteria Screening Marine organisms Sponge Coral Useful materials

项目摘要

Recently, marine microbial consortium has been paid attention as new targets for screening of biologically active agents and so on. However, most of bacteria in environment are unculturable and unidentified. In this study, we aimed to have gene resources and develop the micro-assay system for small number of cells which are directly collected. investigate biodiversity, species identification of bacteria associated with marine sponge and coral, which are well known to produce several biologically active agents. Furthermore, methods for their efficient collection from host cells and genome amplification, and micro-screening system have been developed.Microbial cells were sorted by using flow cytometer and their 16SrDNA analyses were performed before/after cell sorting. 16SrDNA analysis showed that various kinds of bacteria which were not found in the sample not sorted were observed in the sorted cell samples. Those cells are not enough number of cells for construction of genome library. They are subjected to genome amplification by MDA method using phai-29 DNA polymerase. It was confirmed that enough genome DNA was amplified and amplicons less than 10 Kb were not chimeric when the model microbes were used. Furthermore, amine-coated nano-beads showed high performance in DNA extraction/collection.In order to micro-screening of useful materials, several devises were constructed. Cell-capture devise constructed showed 90% cell capture without any non-specific adsorption. Furthermore, micro-capillary devise for micro-screening was also constructed. In the devise, gradient of chemicals introduced into capillary was observed. This showed that it is possible to examine biological effect of target chemicals under the various of their concentration.

近年来，海洋微生物菌群作为生物活性物质筛选等新的靶点受到重视。然而，环境中的大多数细菌都是不可培养和未鉴定的。在这项研究中，我们的目标是拥有基因资源，并开发用于直接收集少量细胞的微量检测系统。调查生物多样性、与海洋海绵和珊瑚有关的细菌的种类鉴定，众所周知，这些细菌会产生几种生物活性物质。此外，还开发了从宿主细胞中高效收集和扩增基因组的方法和微筛选系统。利用流式细胞仪对微生物细胞进行分选，并在细胞分选前后进行16SrDNA分析。16SrDNA分析表明，在分选后的细胞样本中观察到了未分选样本中未发现的各种细菌。这些细胞数量不足以构建基因组文库。用PHAI-29DNA聚合酶的丙二醛(MDA)法进行基因组扩增。结果表明，当使用模式微生物时，扩增出了足够的基因组DNA，且小于10kb的扩增片段不是嵌合的。此外，胺包覆纳米小球在DNA提取/收集方面表现出了高性能。为了对有用的材料进行微筛选，我们构建了几个装置。构建的细胞捕获装置显示90%的细胞捕获没有任何非特异性吸附。此外，还构建了用于微筛选的微毛细管装置。在该装置中，观察到了引入毛细管中的化学物质的梯度。这表明，在不同的浓度下检测目标化合物的生物效应是可能的。