Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA
Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA
复制标题
DOI：
10.1007/s00253-004-1649-8
复制
发表时间：
2004-10-01
影响因子：
5
通讯作者：
Saeki, Y
中科院分区：
文献类型：
--
作者：
Yoshida, N;Saeki, Y
在本研究中，在琼脂暴露过程中，大肠杆菌细胞在外源质粒DNA吸附到温石棉颗粒上转化后表现出耐药性。我们先前证明了在琼脂暴露期间温石棉颗粒对大肠杆菌的渗透。为了进一步研究利用温石棉纤维实现大肠杆菌转化的机制，在琼脂暴露期间，研究了大肠杆菌细胞与温石棉之间的相互作用。用流式细胞术分析温石棉颗粒在温石棉溶液中的分散情况。用搅拌装置将含有表达蓝色荧光蛋白和温石棉颗粒的大肠杆菌细胞的悬浮液暴露在琼脂中，使凝胶表面施加恒定的垂直反作用力。然后使用荧光显微镜来说明荧光素异硫氰酸酯共轭DNA低聚物对温石棉的吸附。当向溶液中加入越来越多的温石棉浓度时，观察到更大的聚集体。随着暴露时间的延长，在此期间，表面水分扩散到琼脂凝胶中，在琼脂表面观察到更高浓度的温石棉。此外，在琼脂表面发育了超过50 μ m的温石棉聚集体。它们的形状像栗子柄。在琼脂暴露过程中，由于在琼脂和搅拌棒的界面上产生的滑动摩擦力，温黏土聚集体穿透了粘附的大肠杆菌细胞的细胞膜。大肠杆菌细胞因此获得了质粒DNA和抗生素耐药性，因为质粒DNA被吸附在温石棉颗粒上。将质粒DNA接种到大肠杆菌细胞中证实了温石棉对大肠杆菌转化的有用性。
In the present study, Escherichia coli cells exhibited antibiotic resistance after transformation with exogenous plasmid DNA adsorbed onto chrysotile particles during agar-exposure. We previously demonstrated penetration of E. coli by chrysotile particles during agar-exposure. To further investigate the mechanism by which transformation of E. coli is achieved through the use of chrysotile fibers, the interaction between E. coli cells and chrysotile was examined during agar-exposure. Dispersion of chrysotile particles within the chrysotile solution was analyzed by flow cytometry. A suspension containing E. coli cells expressing blue fluorescence protein and chrysotile particles was exposed to agar using stirring apparatus, which allowed a constant vertical reaction force to be applied to the surface of the gel. Fluorescence microscopy was then used to illustrate the adsorption of fluorescein isothiocyanate-conjugated DNA oligomers to chrysotile. Larger aggregates were observed when increasing concentrations of chrysotile were added to the solution. With prolonged exposure, during which surface moisture diffused into the agar gel, greater concentrations of chrysotile were observed on the agar surface. In addition, chrysotile aggregates exceeding 50 mum developed on the agar surface. They were shaped like a chestnut bur. The chrysotile aggregates penetrated the cell membranes of adherent E. coli cells during agar-exposure due to sliding friction forces generated at the interface of the agar and the stirring stick. E. coli cells thus acquired plasmid DNA and antibiotic resistance, since the plasmid DNA had been adsorbed onto the chrysotile particles. The inoculation of plasmid DNA into E. coli cells demonstrates the usefulness of chrysotile for E. coli transformation.