Flagella nanotubes visualized microscopically
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
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.
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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