主題主題 ::An improved method for single cell isolation of proAn improved method for single cell isolation of prokaryotes from meso-,thermo- and hyperthermophilikaryotes from meso-,thermo- and hyperthermophili

c environments using micromanipulationc environments using micromanipulation

姓名姓名 :: 黃建章黃建章 學號學號 :93134090:93134090

AbstractAbstract

1.This study presents an improved syste1.This study presents an improved system that enables isolation of single viable m that enables isolation of single viable prokaryotic cell from a mixture of cells.prokaryotic cell from a mixture of cells.

2.The system is based on an inverted mi2.The system is based on an inverted microscope , a microinjector and a micromcroscope , a microinjector and a micromanipulator.anipulator.

所 謂 顯 微 操 作 技 術 是 利 用 牽 引 機 所 謂 顯 微 操 作 技 術 是 利 用 牽 引 機 ( p( puller) uller) 把 細 小 玻 璃 管 把 細 小 玻 璃 管 ( ( 直徑 約 直徑 約 1mm) 1mm) 拉 成 極 細 的 小 管 ， 再 把 此 小 管 磨 成 拉 成 極 細 的 小 管 ， 再 把 此 小 管 磨 成 外 徑 外 徑 88 ～ ～ 10μ m10μ m ， 內徑 ， 內徑 55 ～ ～ 7μ m7μ m 的 注 的 注 射 小 管 射 小 管 ( injection pipet) ( injection pipet) 與 外 徑 與 外 徑 60-8060-80μ mμ m ， 內 徑 ， 內 徑 1010 ～ ～ 15μm15μm 的 固 定 小 管 的 固 定 小 管 ( ( holding pipet) holding pipet) 。 之 後 ， 把 注 射 小 管 。 之 後 ， 把 注 射 小 管 與 固 定 小 管 裝 置在 倒 立 立 體 顯 微 鏡 與 固 定 小 管 裝 置在 倒 立 立 體 顯 微 鏡 的 顯 微 注 射 器 的 顯 微 注 射 器 ( micromanipulator) ( micromanipulator) 上 ，上 ， 在 在 200200 ～～ 400400 倍 的 視 野 下 操 作 。 倍 的 視 野 下 操 作 。

Materials and methodMaterials and method Construction of fluorescent strains for the validation set-up The red fluorescent Pseudomonas putida KT2442∷DsRed was co

nstructed as described by Molbak et al. (2003) and Tolker-Nielsen et al. (2000). The non-conjugative plasmid containing a DsRed gene (Clontech Laboratories, California, USA) was mobilized from E. coli CC118ëpir by the use of helper plasmid RK600 from Escherichia coli HB101. Due to the conjugative properties of RK600, both plasmids conjugate into the non-fluorescent P. putida KT2442. Successful insertion of the DsRed gene into P. putida KT2442 was detected by selective plating on ABT medium (Clark and Maaloe 1967) supplemented with 3 g/l citrate (ABTC) and 50 μg/ml kanamycin. Plates were incubated at 22°C for 48 h, and fluorescence microscopy, in combination with antibiotic selection, was used to verify the presence of successfully inserted red fluorescence.

The green fluorescent E. coli DH5α was constructed by conjugation of the gfp-tagged plasmid pTOL (Kasai et al. 2001) from the donor P. putida KT2442 pTOL to the recipient E. coli DH5α. The conjugation process was carried out after mixing donors and recipients in a ratio of 1:1 on an LB agar plate without antibiotic selection. After incubation for 16 h at 30°C, tenfold dilutions of the mixture was transferred to Luria Bertani (LB) agar with 50 μg/ml kanamycin and incubated at 41°C for 48 h (Molbak et al. 2003). Successful transfer of the plasmid was verified via fluorescent microscopy by streaking out single colonies after 24 h incubation on ABT agar supplemented with 2 g/l glucose (ABTG) and 50 μg/ml kanamycin. Incubation was carried out at 37°C.

tenfold dilutions of the cell mixture of P. putida KT2442∷DsRed and E. coil DH5α harbouring the gfp-labelled pTOL (100:1 ratio) were prepared in sterile PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4), and approximately 0.4 ml was transferred to the sample chamber (Fig. 2).

MicromanipulationMicromanipulation 也有控溫功能也有控溫功能 :: 所以想所以想篩篩

meso-,thermo- and hyperthermophilicmeso-,thermo- and hyperthermophilic 的的菌只要控制好溫度就好了菌只要控制好溫度就好了 (30, 70 or 80°C).

For the solfatara enrichment experiments, 5 μl of the actively growing culture was transferred to a 1:1 mixture of fresh medium and double sterile filtered (0.22 μm) growth medium from the active culture (Fig. 3).

Microscopic images from enrichment cultures prior to single-cell isolation. The picture shows the two enrichment cultures. A. The thermophilic enrichment culture (70°C) from where the Metallosphaera sedula was isolated. B. Enrichment culture from the hyperthermophilic enrichment

(80°C) from where the Sulfolobus solfataricus was isolated

之後之後 PCRPCR 和和 Sequencing,Sequencing, 再做基因庫鑑定再做基因庫鑑定 ,,可得親源關俙．可得親源關俙．