SCREENING OF USEFUL MICROORGANISMS WITH CELL SORTER

使用细胞分选仪筛选有用的微生物

• 批准号: 11650818
• 负责人: KATSURAGI Tohoru
• 金额: $ 1.73万
• 依托单位: NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11650818/
• 关键词: analysis of cells at single-cell level; cell sorting; flow cytometry; microgel beads; astaxanthin; thiamin; pymoloquinoline quinone; microbial production; ゲル微小滴法; Haematococcus pluvialis; 単細胞微細藻類

Microorganisms were screened by flow cytometry for specific characteristics (hyperproduction, in this study) and such organisms were flow cytometrically sorted. Model systems used were :(1) a fluorescent carotenoid astaxanthin (ASX) and a microalga Heamatococcus pluvialis, which produces ASX when encysting,(2) vitamin B1 (B1 ; or thiamin), which is not fluorescent but is chemically transformed to a fluorescent derivative thiochrome, and a sake yeast (Saccharomyces cerevisiae), which produces large amounts of B1, and(3) a coenzyme pyrroloquinoline quinone (PQQ) and two lactic-acid bacteria Acetobacter acetiand Gluconobacter oxidans, which produce PQQ, mainly as holoenzymes (quinoproteins).Hyperproduction of these compounds was induced by treatment with mutagenic compounds and hyperproducers were sorted.Cysts of H.pluvialis were analysed by flow cytometry. The stronger the fluorescence signal was, the larger the size of the cysts was (as microscopic observation), demonstrating sorting of … More H.pluvialis for ASX-hyperproduction. Such cysts were encysted and analysed again to show similar cytographic pattems as those before mutagenesis, suggesting the unstable hyperproducing characteristic obtained.The sake yeast was cultured under various conditions and cells were encapsulated in gel microdroplets (GMDs), typically and successfully with porous glass filter, to produce microcolonies. After thiochrome reaction under certain conditions, epecially with shorter reaction periods and by quick neutralisation of the reaction mixtures, GMDs could be measured for B1 contents with good correlation with the B1 content of the whole cultue (assayed by thiochrome method) and some percentages of GMDs gave regeneration of living cells.The lactic-acid bacteria were cultured under various conditions and cytometrically analysed. Variety of cytogrammes was obtained but without well correlation to the PQQ contents in the whole culture. The UV laser used (625 nm) was not suitable for PQQ because of other compounds which interfered PQQ.Use of arc and deuterium lamps might be considered in further study with PQQ. Less

通过流式细胞术筛选微生物的特定特征（在本研究中为超生产），并对此类微生物进行流式细胞术分选。所用的模型系统是：（1）荧光类胡萝卜素虾青素（ASX）和微藻雨生红球藻（Heamatococcus pluvialis），其在包囊时产生ASX，（2）维生素B1（B1 ;或硫胺素），其不发荧光但化学转化为荧光衍生物硫色素，以及清酒酵母，（酿酒酵母），其产生大量的B1，和（3）辅酶吡咯喹啉醌（PQQ）和两种乳酸菌醋酸杆菌和氧化葡萄糖酸杆菌，其产生PQQ，主要为全酶（醌蛋白）。通过用诱变化合物处理诱导这些化合物的过量产生，并对过量产生者进行分选。荧光信号越强，包囊的尺寸越大（如显微镜观察），表明包囊的分选。 ...更多信息 H.pluvialis的ASX-超生产。这种包囊和分析再次显示出类似的细胞学patterns诱变前，暗示不稳定的hyperproducing characteristics.The清酒酵母培养在各种条件下，细胞被封装在凝胶微滴（GMDs），典型的和成功的多孔玻璃过滤器，以产生小菌落。在一定的条件下，特别是在反应时间较短和反应混合物快速中和的条件下，通过硫色素反应，可以测定B_1含量的GMDs，其与整个培养物的B_1含量（用硫色素法测定）有很好的相关性，并且一定百分比的GMDs可使活细胞再生。结果表明，在整个培养过程中，细胞质的变化是多样的，但与PQQ的含量没有很好的相关性。紫外激光（625 nm）不适用于PQQ，因为其他化合物会干扰PQQ。在进一步研究PQQ时，可以考虑使用弧光和氘灯。少

项目成果

Tohoru KATSURAGI,Satoshi TANAKA,Shin'ya NAGAHIRO,Yoshiki TANI.: "Gel microdroplet technique leaving microorganisms alive for sorting by flow cytometry."Journal of Microbiological Methods.. 42. 81-86 (2000)

Tohoru KATSURAGI、Satoshi TANAKA、Shinya NAGAHIRO、Yoshiki TANI.：“凝胶微滴技术使微生物存活，以便通过流式细胞术进行分选。”微生物学方法杂志.. 42. 81-86 (2000)

Tohoru KATSURAGI,Yoshiki TANI.: "Review : Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting."Journal of Bioscience and Bioengineering. 89. 217-222 (2000)

Tohoru KATSURAGI、Yoshiki TANI.：“综述：通过流式细胞术和单细胞分选筛选具有特定特征的微生物。”生物科学与生物工程杂志。

Tohoru KATSURAGI, Satoshi TANAKA, and Shin'ya NAGAHIRO, Yoshiki TANI: "Gel microdroplet technique leaving microorganisms alive for sorting by flow cytometry."Journal of Microbiological Methods. vol.42. 81-86 (2000)

Tohoru KATSURAGI、Satoshi TANAKA、Shinya NAGAHIRO、Yoshiki TANI：“凝胶微滴技术使微生物存活，以便通过流式细胞术进行分选。”微生物学方法杂志。

Tohoru KATSURAGI,Yoshiki TANI.: "Selection of microorganisms with specific characteristics by single-Cell sorting by flow and microscope-based (including laser scanning) cytometry"Acta Biotechnologica. 21(印刷中(2号)). (2001)

Tohoru KATSURAGI、Yoshiki TANI.：“通过基于流式和显微镜（包括激光扫描）细胞计数的单细胞分选来选择具有特定特征的微生物”Acta Biotechnologica 21（出版中（第 2 期））。

Tohoni KATSURAGI, and Yoshiki TANI: "Review : Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting."Journal of Bioscience and Bioengineering. vol.89. 217-222 (2000)

Tohoni KATSURAGI 和 Yoshiki TANI：“综述：通过流式细胞术和单细胞分选筛选具有特定特征的微生物。”生物科学与生物工程杂志。