Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA.
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 microm 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.
Yoshida
N
N
Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen Kibanadai-Nishi, 889-2192, Japan. a04109u@cc.miyazaki-u.ac.jp
Saeki
Y
Y
D000362:Agar; D017632:Asbestos, Serpentine; D001692:Biological Transport; D004247:DNA / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids; D014169:Transformation, Bacterial
D017632:Asbestos, Serpentine; D008164:Luminescent Proteins; C409504:blue fluorescent protein, Aequorea victoria; D000362:Agar; D004247:DNA