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Flagella nanotubes visualized microscopically

BioPhotonics
Oct 2006
David Shenkenberg

Scientists envision nanotubes as antibiotics, reaction media, catalysts, drug delivery devices and microcircuit conductors. Although carbon nanotubes already exist, they cannot be genetically modified, and their diameters are flexible. In contrast, E. coli flagella nanotubes are natural, they have a fixed diameter, functional groups can be added to them and they can be mass-produced at a low cost. Subra Muralidharan, Brian C. Tripp and colleagues at Western Michigan University in Kalamazoo created flagella nanotubes and analyzed them using fluorescence microscopy, transmission electron microscopy (TEM) and optical trapping.

To synthesize the nanotubes, the researchers made a fusion proteinfrom thioredoxin and a protein subunit of flagella. They inserted peptide-encoding DNA sequences into the thioredoxin active site because previous research had shown that mutated thioredoxin can modify the shape of flagellin. In addition, they mutated flagellin to contain either six or 12 cysteines. Cysteines form disulfide bonds with each other, causing the proteins to fold into various shapes. As a result of these modifications, the flagel-lin-thioredoxin fusion proteins self-assembled into flagella nanotubes. The researchers could separate the nanotubes from the cells only by centrifugation.


Modified flagellin and thioredoxin with a mutated active site self-assembled to form this flagella nanotube.


They performed TEM with a JEOL microscope operated at 300 kV to obtain high-resolution images. They also viewed the flagellin nanotubes using a Nikon epifluorescence microscope with a filter cube that consisted of a 465- to 495-nm excitation bandpass filter and a 515- to 555-nm emission bandpass filter because those wavelengths are compatible with the dye used. They produced an optical trap using an Arryx Nd:YAG laser with a fixed 1064-nm wavelength because the nanotubes do not absorb in that region.

TEM showed that the flagellin nanotubes had bundled together. The scientists demonstrated that these bundles could be disassembled with reducing agents such as dithiothreitol. They observed bundles up to 10 μm long and 200 nm in diameter with an average length of 8 μm.

Fluorescence imaged nanotubes from 4 to 10 μm. While viewing the bundles, they attempted to manipulate the nanotubes with the optical trap, but it did not work, possibly because they used a single well trap. Muralidharan said that they are currently using the optical trap with a spatial light modulator to create up to 200 linear traps.

Since this research has been published, the scientists have successfully created flagella nanotubes in which the flagellin contain modified peptide sequences other than cysteine.

Nano Letters, ASAP edition, Aug. 3, 2006, 10.1021/nl060598u.


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