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

近年来，海洋微生物群落作为生物活性物质筛选的新靶点受到了广泛关注。然而，环境中的大多数细菌是不可培养的和未知的。在本研究中，我们的目标是拥有基因资源，并开发少量直接采集细胞的微量检测系统。调查生物多样性，物种鉴定与海洋海绵和珊瑚有关的细菌，众所周知，它们可以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、基因组扩增和微筛选系统的方法。流式细胞仪对微生物细胞进行分选，分选前后进行16SrDNA分析。16SrDNA分析表明，在分选的细胞样品中发现了未分选样品中未发现的多种细菌。这些细胞数量不足以构建基因组文库。利用phai-29 DNA聚合酶进行MDA方法的基因组扩增。结果表明，当使用模型微生物时，扩增出了足够的基因组DNA，且扩增子小于10 Kb不嵌合。此外，胺包被纳米珠在DNA提取/收集方面表现出较高的性能。为了对有用材料进行微筛选，构建了几种装置。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，观察了进入毛细管的化学物质的梯度。这说明在不同浓度下检测靶化学物的生物学效应是可能的。

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.